the fibroblast and wound repair

Biol. Rev. (1968), 43, pp. 51-96

THE FIBROBLAST AND WOUND REPAIR

BY RUSSELL ROSS Department of Pathology, School of Medicine, University of Washington, Seattle, Washington

(Received 15 May 1967)

CONTENTS

I. Introduction . . . . . Historical review. . . . . Current problems of interest . . Light microscope observations of healing

skinwounds . . . . . 11. The phases of wound repair-electron

microscopy . . . . . The Inflammatoryphase . . .

The cells. . . . . . The granulocyte . . . . The macrophage . . . .

Associated chemical studies . . The proliferative phase . . .

The source of the fibroblast in healing wounds . . . . .

Cytology of the fibroblast . . Histochemical studies . . . Collagen . . . . . .

The macromolecule . . ,

Solubility properties of collagen .

5 1 51 52

54

54

54 54 54 55 56

57

57

58

63 63 63

61

Mucopolysaccharides in healing wounds . . . . .

Chemical observations of healing wounds. . . . . .

The reorganization or remodelling phase Cell turnover . . . . . Turnover of extracellular substances . Collagenase studies . . . .

111. Wound contraction . . . . IV. Factors affecting wound repair . .

Nutrition . . . . . Protein deficiency . . . . Ascorbic acid . . . . . Steroid hormones . . . . Histamine . . . . .

V. Summary . . . . . . VI. References. . . . . . VILAddendum. . . . . .

67

68

69 69 70 70

71

72 72 72 73 77 79 79

81

92

I. INTRODUCTION

The ability to repair wounded tissues is a biologic adaptation, essential to the sur- vival of complex multicellular organisms (Needham, I 964). As a fundamental patho- logic process, the healing wound provides an opportunity to examine in detail many aspects of both the origin and activities of the cells involved in the formation of the substances that comprise the connective tissue. The principal cell is the fibroblast (or its variants, the osteoblast, chondroblast, etc.). Among its known products are collagen, elastic fibres, mucoproteins and the associated protein-polysaccharide complexes of the ground substance.

Historical review Our earliest knowledge of the participation of cells in the process of healing is

derived from the now classical observations of Hunter (1812), Virchow (1852), Cohnheim (1867), and Carrel (1921), to mention but a few. A comprehensive survey of these early investigations can be found in a review by Arey (1936). Arey believed

4-2

52 RUSSELL Ross that cell destruction resulted from either direct trauma or from metabolic deficiencies resulting from injury to the adjacent vasculature. He distinguished between autolysis, due to enzymes elaborated within the injured cells and heterolysis due to destructive enzymes secreted by leucocytes, particularly neutrophils, present in the wound exudate (Opie, 1922; Willstatter, Bamann & Rohdewald, 1930; Stern, 1932).

Carrel (1921) suggested that the products of inflammation and cell lysis have a stimulatory effect upon fibroplasia. His further observations of delayed fibroplasia following an induced neutropenia, and the observations of Bugliari (1927) and Kiaer (1928), who noted that leucocytes and their products, when applied to wounds, stimulated fibroplasia supported this hypothesis. Consequently the concept of a trephone, or wound hormone (Carrel, 1922,1930) as a stimulatory substance elaborated by leucocytes or injured cells has continued to interest many investigators.

Newer investigative techniques have permitted studies of repair from approaches other than those of gross observation and light microscopic analysis. Recent reviews of wound healing (Dunphy & Udupa, 1955; Dunphy, 1958; Edwards & Dunphy, 1958a, b ; Jackson, 1958; Weiss, 1959; McMinn, 1960; Johnson & McMinn, 1960; Dunphy, 1963 ; Chen & Postlethwait, 1964) have emphasized many of the chemical and morphologic characteristics of repair. A correlation of the recently acquired knowledge of cellular and subcellular structure, with observations of the various chemical and physical events that occur during the process of connective tissue repair, can provide further insight into the repair phenomenon.

Current problems of interest Whether the wound is a major defect or a small linear incision primarily apposed by

sutures, the repair process is essentially the same. Initially haemorrhage occurs within the defect and subsequent coagulation produces a fibrin clot containing numerous blood cells (Jorgensen & Borchgrevink, 1964). Small blood vessels that have been injured contract or thrombose, and disappear from the wound area. At later times cells invade the scaffolding of fibrin beneath the scab (see P1.2), apparently using this to aid their movement through the wound, enabling some of them to remove debris while others subsequently synthesize the various connective tissue elements (Chalkley, Algire & Morris, 1946; Weiss & Garber, 1952; Weiss, 1959).

Repair of connective tissue may be arbitrarily divided into three phases. None of these is distinctly separable, since each blends into the next. These may be termed the inflammatory, the proliferative, and the reorganization or remodelling phases of repair. The first two are commonly associated with the formation of granulation tissue; the latter phase is considered to be synonymous with the maturation of scar tissue (Cameron, 1951).

Among the many problems of current interest, the following will be dealt with in this review;

(I) The source or origin of the fibroblast in healing wounds. (2) The various activities of the fibroblast, in particular the synthesis of collagen

and mucopolysaccharides (glycosaminoglycans).

The Jibroblust and wound repair 53 (3) The factors that appear to affect the proliferative phase and the subsequent

(4) The effect of local and systemic factors upon the fibroblasts and their connective remodelling or reorganization within a wound.

tissue products.

fibrin

!ILL- 0 1

PMN leukocyte- ; ’\ I ’,. lymphocyte---

macrophages

capillaries- collagen----

‘ 0 2 4 6 8 10 1213 Days

Text-fig. I. This graph quantifies the elements of the healing wound in the normal guinea-pig as determined from o to 13 days. The various cellular and acellular elements are graded on a o to 3 basis using numbers of cells per high-power field as a basis for grading. These observations were made upon wounds which were prepared for routine light microscopy and stained with haematoxylin and eosin, van Gieson connective tissue stain, phosphotungstic acid- haematoxylin, and Wilder’s reticulin stain. Reprinted by permission of the Rockefeller Institute Press from Ross and Benditt in The Journal of Biophysical and Biochemical Cytology, December 1961, XI, no. 3, p. 679.

54 RUSSELL Ross

Light microscope observations on healing skin wounds Shortly after wounding the skin, the defect is filled with tissue debris, erythrocytes,

leucocytes, fibrin and fluid. In the particular case of a linear skin incision, emigration of leucocytes from vessels occurs within the first 6 hr; their numbers increase to a maximum during the first day, remain at this level until z or 3 days and then decrease. This increase and decrease in granulocytes coincides with an equally prominent rise and fall in the amount of fibrin. The granulocytes are followed into the wound by large mononuclear cells or macrophages which reach their maximum concentration within approximately 48 hr. Lymphocytes are found in large numbers at a somewhat later stage reaching their maximum concentration about the sixth day after wounding. Spindle-shaped cells, identified as fibroblasts", solely on the basis of their shape, can be seen within the wound in the first 4872 hr. They are followed into the wound by capillaries that continue to proliferate until approximately the eighth day. After the seventh or eighth day, the numbers of fibroblasts and blood vessels begin to decrease, and appear to become constant by the fourteenth day. Collagen fibres may be detected by the light microscope within the intercellular spaces after approximately 4 days, and continue to increase in both number and size for several weeks when the formation of a scar has occurred. The length of each of these phases is dependent upon the size of the defect. Connective tissue formation proceeds more rapidly near the wound margin, whereas the central area, particularly in larger wounds, is generally the last to heal. These sequences were demonstrated in a quantitative histologic study of cells and extracellular elements in the guinea-pig (Ross & Benditt, 1961). The results are graphically presented in Text-fig. I.

11. THE PHASES OF WOUND REPAIR-ELECTRON MICROSCOPY

The injammatory phase The cells

The granulocyte An optimal inflammatory response has long been considered to be a pre-requisite for

an orderly sequence of fibroplasia in healing wounds. Yet this relationship has been studied by relatively few investigators, and its role in wound healing is poorly understood.

The early cell reaction following wounding was attributed to a period of ' defence and demolition' (Needham, I 952). Neutrophilic granulocytes are the most prominent cells seen during the first 12-16 hr. (Raekallio, 1961 ; Ross & Benditt, 1961). The neutrophil contains cytoplasmic granules of varying size and density, known to be repositories of acid hydrolytic enzymes (Cohn & Hirsch, 1960), and have been considered to be a form of lysosome (de Duve, 1959, 1963), important in the process of phagocytosis (Pl. I). The lysosomal enzymes may play an equally influential extracellular role in

* For purposes of discussion, a fibroblast shall be considered to be a cell either actively engaged or potentially active, in terms of its organelle development, in the synthesis of collagen. A fibrocyte is considered to be a resting or inactive fibroblast.

The fibroblast and wound repair 55 wounds as a result of neutrophil disruption and release of enzymes from the granules into the surrounding environment.

In the healing wound the major role of the granulocyte does not appear to be only that of phagocytosis. Large numbers of degenerated neutrophils can be seen during the first 24 hr. Many granules, morphologically similar to those in intact neutrophils, are found in both the intercellular spaces and in various phases of degradation within phagocytic vacuoles in macrophages (Pls. I and 2). The lysis of neutrophils in a wound, with resultant release of their stores of hydrolytic enzymes could represent an important function of this cell. The presence of such enzymes in the extracellular compartment could facilitate the breakdown of extracellular components, such as fibrin or tissue debris, and thus aid in their removal from sites of injury. Consistent with this idea are observations of areas of decreased density surrounding neutrophil granules deposited in fibrin and extracellular debris (Pl. 2). Sites such as these may represent lysis of the fibrin, or debris, prior to its removal by macrophages.

Further evidence for fibrinolytic activity mediated by granulocytes was presented by Barnhart (1965) and Riddle & Barnhart (1964, 1965). They studied fibrinolysis in a combined fluorescence and electron microscopic study of dog skin windows and observed what they interpreted as phagocytosis and intracellular digestion of fibrin, as well as extracellular lysis of fibrin by free neutrophil granules.

The activities of the neutrophil in phagocytosis and in granule release seem to be well established. The question still remains as to whether these phenomena serve as a stimulus for fibroplasia as many investigators have suggested. It has been shown by Carrel (1921) that a protected open wound was minimally inflamed, and healed very slowly when there was no antecedent trauma. Selye (1953) emphasized that the amount of fibroplasia in the rat granuloma pouch was dependent upon the character of the irritant that was introduced. Those irritants that evoked a marked exudation of neutrophilic leucocytes were followed by rapid fibroplasia ; whereas, those that evoked a minimal exudation displayed a delay, both in the appearance and amount of connective tissue formed. Other substances such as carrageenin (a polysulphated galactose) (Williams, 1957) also induce a fibroblastic response preceded by marked inflammation, consisting of both granulocytes and large mononuclear, phagocytic cells. If the inflammatory response is depressed by agents such as cortisone, a subject to be discussed in more detail in a later section, then fibroplasia is either decreased or delayed in onset (Spain, Molomut & Haber, 1952; Savlov & Dunphy, 1954; Pernokas, Edwards & Dunphy, 1957). It would appear, therefore, that the initial inflammatory reaction does bear a relationship to fibroblast proliferation and to the synthesis of connective tissue (Edwards, Pernokas & Dunphy, 1957; Schilling, Favata & Radako- vich, 1953 ; Williams, 1957). The nature of this relationship remains to be more clearly elucidated.

The Macrophage Source. Besides the granulocyte, the other prominent cell of the inflammatory re-

sponse is the large mononuclear cell or macrophage. The site of origin of these cells has been said to be either the adjacent tissues, or the blood, or both. In a series of

56 RUSSELL Ross studies, utilizing th~midine-~H in parabiotic rats, Volkman & Gowans (1965 a) used the skin-window technique to examine the cell response in a non-immunologic, inflammatory reaction. They demonstrated that monocytes circulating in the blood and subsequently found in the windows were derived from a relatively small, rapidly dividing, pool of precursors. Volkman & Gowans (1965 b) further determined that these cells were derived from marrow, rather than lymphoid tissues, by irradiating the lymphoid tissues and shielding the marrow, or by using labelled cells derived from lymph nodes or marrow that they administered intravenously. These important investigations demonstrated that the principal phagocytic cell in the healing wound, like the cells in the skin windows, arrives via the blood. The role played by the resident tissue cells, often called histiocytes, appears to be minimal under these circumstances.

Morphologic characteristics. Prior to becoming actively phagocytic, the macrophage can be recognized and distinguished by virtue of its fine structure characteristics (Palade & Porter, 1954; Palade, 1955; Ross & Benditt, 1961; de Petris, Karlsbad & Pernis, 1962; Gieseking, 1963; Ross & Lillywhite, 1965). These include a fairly extensive network of smooth endoplasmic reticulum, a sparse, poorly developed rough endoplasmic reticulum, numerous randomly dispersed, intracytoplasmic filaments (ca. 50-80A. in diameter) and many vacuoles of varying size and density (Pl. 2).

These vacuoles often contain whorls of membranous material together with ingested cell debris.

The most striking morphological difference between the macrophage (or potential macrophage) and the fibroblast consists in the disparity in the development of the rough endoplasmic reticulum in these two cell types. The membranes of rough endoplasmic reticulum of the monocyte (or macrophage) contain relatively few sparsely distributed, attached ribosomes, whereas those in the fibroblast contain very few vacant sites (Pls. 2-4). Once the monocyte has become actively phagocytic it is more easily recognized. Fibroblasts, like numerous other cells, are also capable of phagocytosis, but can be distinguished by these differences in fine structure, whereas it would be extremely difficult to distinguish between these two cell types by routine light microscopic procedures. Observations of these cells by time lapse cinemato- graphy might provide further information.

Associated chemical studies DNA is found in appreciable amounts whereas RNA is found in relatively small

amounts during the inflammatory phase of healing. This has been ascribed to the presence of large numbers of blood leucocytes in the wounds (Woessner & Boucek, 1961 a ; Williamson & Guschlbauer, 1961 ; Viljanto, 1964). Such observations would be anticipated since the mature granulocytic celI series is not active in protein synthesis and contains little, if any, cytoplasmic RNA (Hirsch, 1965).

A large amount of PAS-positive, extracellular material is present in wounds during the first three to four days after wounding. A sizeable quantity of hexosamine is also present at this same time (Dunphy & Udupa, 1955; Viljanto, 1964). The bulk of this hexosamine appears to be due to the cleavage of plasma glycoproteins (Grillo, Watts & Gross, 1958; Jackson, Flickinger & Dunphy 1960), and is undoubtedly related to the

The fibroblast and wound repair 57 increased metachromasia in the extracellular compartment described in early wounds (Dunphy & Udupa, 1955). The mucoprotein content of granulation tissue represents a minor proportion of the hexosamine content of this tissue (Bentley, 1967).

During the first few days there is no collagen present and a network of fibrin initially unites the wound margins and also provides a scaffolding for the migratory cells that enter into the wound.

The proliferative phase The source of the fibroblast in healing wounds

The source of the connective tissue-forming cell in healing wounds has been a matter for debate since Cohnheim ( I 867) suggested that cells from the blood may serve as progenitors for fibroblasts. This same notion has since been proposed by many investigators (Fischer, 1925 ; Maximow, 1927; Carrel & Ebeling, 1926; Bloom, 1927; Maximow, 1928; Moen, 1935; Allgower, 1956; Allgower & Hulliger, 1960; Petrakis, Davis & Lucia, 1961; Petrakis, 1961). Most attempts to study this problem have utilized modifications of various techniques of tissue culture, or examination of blood cells, usually obtained from the buffy coat, grown in subcutaneously implanted Millipore filter diffusion chambers. Several investigators have examined this problem in vivo by utilizing tritiated thymidine (MacDonald, 1959; Grillo & Potsaid, 1961 ; Grillo, 1963). These investigators have been unable to verify a haematogenous origin for the fibroblast.

By irradiating wounds locally, 28 hr. after wounding, Grillo (1963) demonstrated a 50 % reduction in the mesenchymal cell proliferation. He could produce the same effect by irradiating the wounds as early as 20 min. after wounding. At 20 min. the granulocytic leukocytes represent the majority of the cell population in the wounds. Since the neutrophil is unlikely to serve as a source of fibroblasts, it would appear that the mesenchymal cells located in areas adjacent to the wound, that were susceptible to radiation, were the source of the fibroblasts, In addition, the principle cells to demonstrate th~midine-~H uptake were perivascular cells from the loose connective tissue of the wound margins. These observations and those by Gliicksmann (1964) in studies using colchicine to arrest cell mitosis, support the notion that wound fibroblasts are derived from the adjacent connective tissues.

In contrast, a series of experiments to determine the ability of blood cells to trans- form into fibroblasts were performed by Petrakis et al. (1961). These investigators used blood withdrawn by either cardiac puncture, or venipuncture, to prepare buffy coat that was subsequently placed in diffusion chambers. They found fibroblasts and collagen in these chambers after 4 weeks of subcutaneous implantation. Investiga- tions similar to those of Petrakis et al. (1961) were performed in an attempt to rule out the possibility of contamination of the buffy coat by connective tissue cells (Ross & Lillywhite, 1965). Ross & Lillywhite used buffy coat obtained both by cardiac puncture and venipuncture as well as by arterial and venous cannulation. They could demonstrate no collagen formation in chambers containing buffy coat from cannulated blood. Hence it must be supposed that the earlier results (Petrakis et al. 1961) were caused by contamination of the buffy coat by cells picked up during cardiac puncture

58 RUSSELL Ross or venipuncture. It has not been possible to verify the hypothesis that the haematogenous leucocyte serves as a precursor for the fibroblast in healing wounds. The present evidence indicates that fibroblasts present in a wound are derived from the surrounding connective tissue, perhaps from perivascular mesenchymal cells.

Cytology of the fibroblast The fibroblast has been extensively examined by numerous investigators, both in

healing wounds and in other situations where relatively rapid connective tissue formation occurs, such as the developing embryo and tissue culture (Porter, 1953; Wassermann, 1954; Palade, 1956; Palade & Porter, 1954; Fitton Jackson, 1956; Wassermann & Kubota, 1956; Palade, 1 9 5 8 ~ ; Porter & Pappas, 1959; Gieseking, 1959a, 6 ; Kajikawa, Tanii & Hirono, 1959; Chapman, 1961 ; Merker, 1961 ; Peach, Williams & Chapman, 1961; Ross & Benditt, 1961; Movat & Fernando, 1962; Fernando & Movat, 1963; Jorgensen, 1963b; Cliff, 1963; Goldberg & Green, 1964; Ross, 1964; Ross & Benditt, 1964).

The term fibroblast is used for the cell actively engaged in the production of connective tissue matrix, in contrast to its dormant phase as a fibrocyte. The fibroblast can be recognized and classified on the basis of the following fine structure characteristics :

(a) An abundant, extensive rough endoplasmic reticulum, the cisternae of which are often arranged in long, intercommunicating parallel sacs (Pls. 4, 5) . Characteristic- ally, large groups of ribosomes are attached to the rough endoplasmic reticulum membranes and take the form of curved rows or pairs of rows (Pls. 4, 6).

(b ) A prominent Golgi zone, with groups of saccules and vesicles apparently randomly located throughout the cell (Pl. 7).

(c ) Large mitochondria with irregular cristae (Pls. 4, 5) . ( d ) Aggregates of fine filaments (approximately 50-80 A in diameter), vesicles and

caveolae located peripherally within the cytoplasm (Pls. 4, 5 ) . (e ) A large nucleus with one or more prominent nucleoli (PI. 5 ) . (f) Occasional dense cytoplasmic bodies (Pls. 4-6). The rough endoplasmic reticulum. The most distinctive feature of the fibroblast is the

extensive, highly developed rough endoplasmic reticulum. This organelle takes the form of a three-dimensional network of flat or dilated sacs or channels that occupy approximately 35 % of the volume of the cell (Ross & Benditt, 1965). This ribosome- studded canalicular system, now clearly associated with cells that synthesize and secrete extracellular protein, is randomly distributed throughout the fibroblast cytoplasm (Pls. 4, 5 and 7). This is in contrast with other secretory cells such as those of the exocrine pancreas where the organelle is found in a specific region in the cell.

When the membranes of the rough endoplasmic reticulum are tangentially sectioned, the attached ribosomes are seen to be arranged in rather large aggregates that have characteristic patterns (Pl. 6). It has been suggested that these aggregates are polyribosomes; however it is not clear what role, if any, the membranes to which they are attached play in the formation of these patterns.

It is relatively common to find sites in the cortical cytoplasm of the fibroblast where projections of the rough endoplasmic reticulum cisternae come very close to the surface

The jibroblast and wound repair 59 of the cell (Pl. 5-8). In some regions there appears to be intimate contact, or communi- cation, between the cisternae of the ergastoplasm and the exterior of the cell (Karrer, 1960; Ross & Benditt, 1964, 1965) (Pl. 8). These may represent sites where material present in the ergastoplasmic cisternae may be released into the extracellular space. This suggestion is discussed more fully in the section on secretion of collagen precursors.

The Go@ apparatus. The fibroblast is similar to other secretory cells in that it has a well developed Golgi apparatus (Pl. 7) . In contrast to many secretory cells, however, this membrane complex is randomly dispersed throughout the cytoplasm of the fibroblast and occupies approximately 9 % of the cell volume (Ross & Benditt, 1965). Most fine structure studies of protein synthesis and secretion have dealt with glandular cells, such as those of the pancreatic acini. These cells are polarized and usually release their products from their apex. They are characterized by a juxtanuclear Golgi zone that is apical to the nucleus. In contrast, the fibroblast is a motile cell with numerous cytoplasmic extensions, and it can probably secrete its products from any surface. These differences may partially explain why the Golgi zone of the fibroblast is randomly dispersed throughout this cell.

Densely staining material has been reported within Golgi saccules in fibroblasts in regenerating tendon (Fernando & Movat, 1963). Similar material can be seen in the Golgi vesicles in PI. 7 . Sheldon & Kimball (1962) described banded material within the Golgi complex of papain-treated chondrocytes that they interpreted to be collagen. These observations have not been confirmed in the numerous reports that have since appeared and there is no evidence that the vacuoles containing banded material are part of the Golgi complex. Sites, interpretable as communications of cisternae of rough endoplasmic reticulum with Golgi cisternae, similar to those in other cell types, can also be seen in the fibroblast (Pl. 7) . Coated or fuzzy vesicles are also commonly seen in association with the Golgi complex.

The Golgi zone has been associated with carbohydrate synthesis (Peterson & Leblond, 1964; Fewer, Threadgold & Sheldon, 1964; Godman & Lane, 1964; Neutra & Leblond, 1966) and with the ‘packaging’ of various substances with proteins and enzymes synthesized in the rough endoplasmic reticulum (Caro & Palade, 1964; Jamieson & Palade, 1966). Its role, if any, in the synthesis of collagen is less clear, and will be discussed later in this review.

The mitochondria. The mitochondria of the fibroblast are characteristically large, and contain numerous cristae. In contrast to the mononuclear cells seen in healing wounds, the mitochondria1 cristae of the fibroblast are irregular in length and shape, the matrices of the mitochondria may appear pale, and the mitochondria may have a ‘swollen’ appearance (PI. 4) depending upon the mode of fixation.

Nucleus. The nucleus of the fibroblast is characteristically large, and ovoid, and contains one or more prominent nucleoli (Pl. 5 ) .

Other cytoplasmic components. Other cytoplasmic components less frequently seen in the fibroblast include an occasional dense body (Pls. 5 , 6), lipid droplets (Pl. 5), and multivesicular bodies (Pl. 6).

Cytoplasmic $laments. Porter & Pappas (1959) described zones, adjacent to the cell

60 RUSSELL Ross surface, in chick embryo fibroblasts, where closely packed filaments provide areas of increased cortical density. They observed small collagen fibrils in the extracellular regions near these densities (Pls. 4,s)) and considered these dense regions to be possible sites where extracellular collagen precursors were aggregated into fibrils, possibly by ecdysis, or cytoplasmic shedding. Other investigators (Yardley, Heaton, Gaines & Shulman, 1960; Chapman, 1961 ; Cameron, 1961 ; Porter, 1964) have also described regions in the fibroblast where these intracytoplasmic filaments appear to ‘merge ’ with the cell surface and ‘pass out’ of the cell. Ross & Benditt (1961, 1964) felt that these sites represented regions where the cell surface had been tangentially sectioned, resulting in an apparent continuity between the filaments and the extracellular regions (Fig. 5). Goldberg & Green (1964) drew similar conclusions from their observations of tissue culture fibroblasts. By examining serial sections of fibroblasts in both longitudi- nal and transverse sections, they were able to demonstrate that these filaments lay either entirely within or outside of the cell. Similar observations were made in collagen forming cells grown in diffusion chambers (Ross & Lillywhite, 1965).

Small extracellular filaments are characteristically seen immediately adjacent to the surface of wound fibroblasts. They are commonly positioned close to the cortical aggregates of intracellular filaments (Pls. 4, 5) . Tangential sections of such regions would provide a misleading picture of continuity between intra- and extracellular filaments. Such a situation is illustrated in P1. 4.

Individual filaments (50-80 A in diameter) can also be seen randomly dispersed throughout the cytoplasm of fibroblasts. These may be related to cell motility, or contractility, and are probably similar to those seen in other motile cells such as the monocyte (de Petris et al. 1962).

Periodic banding in collagen. Appropriate calculations demonstrate that in collagen fibrils of less than IOO A. diameter it may not be possible to demonstrate the characteristic periodic 600-700 A. banding (Ross & Benditt, 1961). Other criteria would then be necessary to recognize these fibrils as collagen. Therefore, fine, extracellular filaments seen in wounds may represent either early aggregations of collagen precursors into fibrils, whose diameter is too small to display the characteristic banding, or they may be other extracellular proteins.

The mode of fibril formation. The site and sequence of collagen fibril formation has been debated for many years. Schwann (1847), Remak (1852), and Schultze (1861) felt that the fibres developed by ecdysis, or shedding of peripheral cytoplasm. A similar interpretation has been given to both the fine structure observations of Porter & Pappas (1959), and to the observations of Stearns (194oa, b). In her series of now classical experiments, Stearns studied collagen formation by fibroblasts, directly observable in the rabbit ear chamber. She noted cellular projections, or blebs, that she interpreted to be cytoplasmic shedding. Stearns felt that this represented the mode of fibril release by the cells, and observed that cellular orientation influenced fibre arrangement. A similar interpretation was drawn by Yardley et al. (1960) and Chapman (1961) in fine structure studies of tissue culture and the carrageenin granuloma respectively.

A second school of thought developed from the observations of Virchow (1852) and

The jibroblast and wound repair 61 Kolliker (1861) who felt that cells secreted either the fibrils or their precursors. It has been demonstrated that collagen fibrils can form from soluble precursors independent of cells (Gross, Highberger & Schmitt, 1955; Hodge, Highberger, Deffner & Schmitt, 1960). In their studies of healing wounds, Ross & Benditt (1961) noted that as a wound gets older, at least two separate populations of fibril size were present in the extracellular spaces. Both of these populations showed an increase in fibril diameter with increasing age, and the smaller fibrils were generally found closer to the cells. In addition, as mentioned previously, connective tissue cells contain peripheral vesicles and sites of approximation of ergastoplasmic cisternae with the plasma membrane (Revel & Hay, 1963; Ross & Benditt, 1964, 1965; Goldberg & Green, 1964). Both represent possible sites of transfer of collagen precursors to the cell exterior, where aggregation and fibril formation can occur. In addition, autoradiographic studies (Ross & Benditt, 1962b) indicate that wound fibroblasts continually synthesize and secrete collagen.

The present evidence supports the notion that the cell secretes collagen precursors into the extracellular region where they aggregate into new fibrils, or join pre-existing fibrils whose diameter is thereby increased. According to this interpretation, the smaller fibrils would in general have been formed more recently than those of larger diameter (Jackson & Bentley, 1960; Vogel, 1964). It is probable that fibril formation is dependent upon some form of cellular control of the local environment that permits aggregation of precursors into fibrils.

Histochemical studies Enzymes. Most histochemical investigations of wounds have utilized determinative

approaches to demonstrate substances such as mucopolysaccharides, while more recent studies have examined the presence of various enzymes in the cells of healing wounds. The earliest attention was paid to alkaline phosphatase (Fell & Danielli, 1943; Danielli, Fell & Kodicek, 1946; Bunting & White, 1950). This enzyme was originally said to be present in healing wounds in polymorphonuclear neutrophilic leucocytes, fibroblasts, and regenerating epithelium. With improvements in techniques of enzyme localization, French & Benditt ( I 954) demonstrated that the alkaline phosphatase present in regenerating skin wounds is primarily associated with the developing epithelium of hair follicles. They concluded that there was non-selective absorption of the enzyme by collagen fibres, as had been previously demonstrated by Gold & Gould (1951). Gold & Gould could find no effect on collagen formation by either the direct application of alkaline phosphatase, or its inhibitors, in suitable experimental conditions. Furthermore, Johnson & McMinn (1958) were unable to demonstrate the presence of this enzyme in granulation tissue from healing skin wounds in the cat.

Another enzyme, leucine amidase, has been demonstrated both in the connective tissue surrounding neoplasms, and in inflammatory tissues (Monis, Nachlas & Seligman, 1959). This enzyme was localized in the cytoplasm of fibroblasts in both these studies, where its presence was considered to be ‘an inherent property of fibroblastic activity ’. In a subsequent study, Monis (1963) demonstrated aminopeptidase

62 RUSSELL Ross in wound fibroblasts and felt that this histochemical reaction was a direct simple test for the demonstration of the distribution of fibroblasts in many situations. He showed that this enzyme was primarily present during the phase of proliferation and was therefore related in some way to this activity. The presence of aminopeptidase has also been correlated with a fibro-proliferative response to invading tumours in experimental animals (Mottet, 1961).

In an extensive series of studies Raekallio et aE. (1964) have reviewed earlier investigations of wounds (Raekallio, 1960, 1963 a, b ; Raekallio & Levonen, 1963 a-c) in which the presence of numerous enzymes, including the non-specific phosphatases, ATPase, esterases, beta glucuronidase, aminopeptidase, cytochrome oxidase, mon- amine oxidase, and succinic dehydrogenase were described. They observed that the intensity of the histochemical reactions of all these enzymes was less at the wound periphery, and greater in the centre of the wounds during the first 24-48 hr. after wounding.

Those enzymes that have been associated with lysosomes (de Duve, 1959, 1963) and demonstrated in granulocytes (Cohn & Hirsch, 1960; Hirsch, 1965) would be expected to be present during the inflammatory phase.

Several oxidative enzymes were found to be present in healing fractures (Balogh & Hajek, 1965). Their activity was high during the proliferative phases of both osteogenesis and chondrogenesis. As cell maturation and healing proceeded, the amount of the oxidative enzymes appeared to decrease, although their presence at a relatively low level was still detectable.

These histochemical observations are of value in determining the cellular localization of the enzymes in question, but reveal little information concerning the roles of these enzymes in the functional activities of the cells involved.

Mucopolysaccharides. Several investigators have examined wounds for the presence of mucopolysaccharides and mucoprotein-containing granules within connective tissue cells. These studies indicated the presence of such granules in wound fibroblasts (Gersh & Catchpole, 1949; Penney & Balfour, 1949; Fitton Jackson, 1955). Fibro- blast granules were characterized by Fitton Jackson (1955) on the basis of their positive staining with the periodic acid-Schiff technique and the Hale colloidal iron method, and their chromotropism after staining with toluidine blue. In addition, when examined by ultraviolet microscopy, the granules absorbed at 280 mp and not at 3 13 mp, indicating that they contained protein. It is interesting, however, that electron microscopic observations of the fibroblast do not show it to be a granule-rich cell (Ross & Benditt, 1961, 1965). It is difficult to find a cytoplasmic structure that corresponds to the granules observed with the light microscope other than the ergastoplasmic cisternae or Golgi complex. The association of the Golgi complex with polysaccharide synthesis and the multiplicity of Golgi complexes in the fibroblast suggest that this organelle may be responsible for Fitton Jackson’s observations.

Mucopolysaccharides have also been detected in fibroblasts by sulphation metachromasia (Windrum, 1958), although with this technique they do not appear to be present in the form of granules. Dunphy & Udupa (1955) also examined healing wounds for the presence of mucopolysaccharides and collagen. They found a marked

The jibroblast and wound repair 63 increase in the amount of mucopolysaccharide as measured chemically up to the first eight days of healing, after which there was an apparent decrease.

Collagen The macromolecule

Collagen has a unique amino acid composition, as demonstrated by the fact that the molecule contains exceptionally large amounts of glycine (c. 30 yo), proline and hydroxyproline (c. 12 % each), and substantial amounts of both glutamic and aspartic acids. In vertebrates, hydroxyproline and hydroxylysine appear to be unique to collagen' with the exception of the small amount of hydroxyproline present in elastin. In addition, collagen contains no tryptophane or cystine, and very small amounts of tyrosine and methionine (Bowes & Kenten, 1948; Gustavson, 1956; Eastoe, 1955; Harkness, 1955; Gross, Dumsha & Glazer, 1958; Piez & Gross, 1959; Piez, 1960). Hydroxyproline is commonly used as a means of identifying the presence of collagen in tissues (Neuman & Logan, 1949, 1950; Martin & Axelrod, 1953). The constituent polypetpide chains of this protein are joined together in the form of three separate alpha helices. These are wound about each other in a helical fashion and are apparently held together by covalent bonds resulting from aldehyde conversion of adjacent lysine molecules (Bornstein, Kang & Piez 1966) as well as other chemical bonds to form the tropocollagen macromolecule. Each of the individual polypeptide chains has been named an alpha chain, two of which were thought to be identical, and the third different (Piez, Lewis, Martin & Gross, 1961 ; Piez, Eigner & Lewis, 1963). However, in one species (codfish skin collagen) each alpha chain has been shown to have a different amino acid composition (Piez, 1964). This may well be found to be the case in other forms of collagen as well.

In solution, the tropocollagen macromolecule appears as a cylindrical unit with a diameter of 14A., a length of approximately 3000A., and a molecular weight of approximately 300,000. Classical fibrillar structures result from the aggregation of these macromolecules. These fibrils demonstrate a periodic banding, after appropriate staining, with major periods approximately 600-700 A apart, between which numerous intra-period bands can be found. The areas of banding appear to be related to regions in the fibril where accumulations of polar amino acids give these segments greater affinity for the various anionic and cationic dyes that are used in staining collagen. These bands enable ready identification of collagen fibrils with the electron microscope.

Extensive reviews of the composition and structure of collagen can be found in the text edited by Ramanathan (1962), and in papers by Bear (1952), Harrington & von Hippel (1961), Harkness (1961), and Ramachandran (1963).

Solubility properties of collagen

formation and increasing stability with age. Collagen has interesting solubility properties that provide further insight into its

An investigation by Jackson & Bentley (1960) on the significance of the extractable

64 RUSSELL Ross collagens demonstrated that there was a continuous spectrum of aggregates of collagen molecules that varied in their degree of cross-linkage, depending upon the time elapsed after their release into the extracellular environment. According to these investigators, the various media in which they were extractable demonstrated the relative amount of intermolecular bonding, and therefore coincided with the length of time between secretion and fibril formation. The amino acid composition of each of these fractions of collagen appeared to be essentially the same, and it was possible to obtain native type fibrils from each of these solutions after appropriate treatment. Therefore, it is reasonable to assume that after collagen synthesis and release into the extracellular milieu, the various stages of collagen solubility represent a spectrum of maturity in terms of cross-linkage and bonding between the constituent parts of the molecules and the fibrils.

Collagen biosynthesis. In I 949 Stetten demonstrated that hydroxyproline was not utilized in the synthesis of collagen. I t was further known (Stetten & Schoenheimer, 1944) that proline served as the precursor for both proline and hydroxyproline in this protein. The same appears to be true for the conversion of lysine to hydroxylysine (Sinex & Van Slyke, 1955; Piez & Likins, 1957; Sinex, Van Slyke & Christman, 1959). The chemical site where hydroxylation of proline andlor lysine occurs during collagen synthesis within the fibroblast remains unsettled. This hydroxylation step is known to be missing in scurvy and therefore several investigators looked for the presence of a proline-rich, hydroxyproline-poor precursor during scurvy (Robertson & Schwartz, 1953; Gould & Woessner, 1957). No evidence was found for such a precursor within the extracellular material from scorbutic wounds (Robertson, Hiwett & Herman, 1959; Gross, 1959). Several studies (Manner & Gould, 1962, 1963; Coronado, Mardones & Allende, 1963; Jackson, Watkins & Winkler, 1964; Urivetzky, Frei & Meilman, 1965) have identified an s-RNA-hydroxyproline and s-RNA-hydroxylysine within chick embryo material. In contrast, however, Peterkofsky & Udenfriend (1961,1963,1965) presented evidence in cell-free systems, derived from chick embryos, that hydroxylation occurred in small peptides in the microsomal fractions. In addition, Juva & Prockop (1964) found that puromycin inhibited the synthesis of collagen hydroxyproline from free proline to a greater extent than it inhibited the incorporation of proline into polypeptides. They interpreted this to indicate that puromycin inter- rupted the synthesis of a proline-rich polypeptide precursor of collagen that served as a substrate for the hydroxylation of proline located at specific sites in the peptide. Prockop & Juva (1965) also prepared an intermediate for hydroxyproline synthesis, from chick embryos, that appeared to be a peptide of considerable size. Other investigators have arrived at similar conclusions (Konno & Tetsuka, 1962; Lukens, 1965). It appears, therefore, that hydroxylation of proline takes place after peptide formation has occurred.

There is little doubt that hydroxylation is an intracellular phenomenon. The form, however, in which the collagen molecule leaves the cell remains to be established. At the moment there seem to be at least two reasonable possibilities: (I) that the three individual alpha chains are synthesized and aggregated into tropocollagen macromolecules within the cell, prior to their release into the extracellular environment

The Jibroblast and wound repair 65 where they aggregate into fibrils: (2) that the three individual alpha chains are synthesized within the cells in smaller units and individually released into the extracellular environment where aggregation, directed by the sequence of amino acids within the units, first forms tropocollagen macromolecules, followed by further aggregation into collagen fibrils.

Organelles associated with collagen synthesis and secretion. The now classical investigations of protein synthesis, in fractions of liver and pancreas, by Palade & Siekevitz (1956 a, b) demonstrated that a well-developed rough endoplasmic reticulum, with its characteristic aggregates of ribosomes, is the counterpart of the microsomes derived from cell fractionation (Palade, 1958 b). This organelle is now commonly associated with cells that synthesize protein for export (Porter, 1961). Lowther, Green & Chap- man (1961) examined the carrageenin granuloma from the guinea-pig after cell fractionation and separation of the various subcellular components. In their studies, the primary cell fraction associated with tritiated proline incorporation, during collagen synthesis in vivo, was the microsomal fraction. Just as in the liver and pancreas, they found that the ribosomes of the endoplasmic reticulum of the intact fibroblast represented the principal site of protein synthesis.

Autoradiography at the light, and particularly, the electron microscopic level using proline-3H, allows one to study the participation in protein synthesis of cell organelles that are less easily separable by cell fractionation (Ross & Benditt, 19626, 1965). By light microscopy it was seen that proline was maximally incorporated into the fibroblast of the wound within I hr. after intraperitoneal administration. Later, and concomitant with its disappearance from the fibroblasts, an increased amount of proline appeared extracellularly in collagen.

Qualitative evaluation of electron microscope autoradiographs of this phenomenon demonstrated that the first cell organelle to be maximally labelled was the endoplasmic reticulum. Subsequently pr~l ine-~H reached a maximum in the Golgi apparatus. The peripheral cytoplasmic components, such as vesicles, were maximally labelled after the ergastoplasm and Golgi, and prior to the extracellular collagen fibrils (Pls. 9, 10). In contrast, a quantitative analysis of these results (Ross & Benditt, 1965) demonstrated that the shape of the curves of concentration of label was not consistent with passage of all of the label from the ergastoplasm, to the Golgi complex and thence to peripheral components, and finally out of the cell. This sequence has been qualitatively demonstrated for the chondroblast (Revel & Hay, 1963) during collagen formation, and quantitatively for the uptake of leucine by the pancreatic acinar cell (Caro & Palade, 1964; Jamieson & Palade, 1966), during the synthesis of zymogen granule protein. The pancreatic acinar cell presents an entirely different problem of analysis from the fibroblast, since the pancreatic cell is polarized, and much of its secretory protein is stored in zymogen granules, containing both protein and polysaccharide, before it is secreted. The data of Ross & Benditt (1965) suggested the possibility that after collagen had been synthesized in the endoplasmic reticulum the collagen precursors were secreted directly from this subcellular system into the extracellular space.

This proposal by Ross & Benditt (1965) leaves the role played by the Golgi complex of the fibroblast to be clarified. Autoradiographic studies of the utilization of pr~l ine-~H

5 Biol. Rev. 43

66 RUSSELL Ross have demonstrated that some of this amino acid passes through the Golgi apparatus in wound fibroblasts (Ross & Benditt, 1965). As stated earlier, amino acids present in the Golgi apparatus appear to be in proteins other than collagen that were synthesized in the ergastoplasm and then passed to the Golgi apparatus prior to being secreted into the ground substance. The Golgi complex has been implicated as the site of polysaccharide synthesis. Observations that support this contention include the localization of 36s-labelled sodium sulphate in the Golgi saccules of wound fibroblasts (Pl. I I)

(R. Ross, unpublished observations) and tissue culture fibroblasts (G. C. Godman,

C--, amino acids 4 collagen precursors I+ protein-polysaccharide complexes

Text-fig. 2. The cell in this figure represents an idealized diagram of a fibroblast. Two postulated pathways of amino acid incorporation into protein in this cell are shown by the three types of arrow. The arrow attached to the black dot represents the entrance of amino acids, presumably through the cell membrane, to the aggregates of ribosomes attached to the rough endoplasmic reticulum. There the amino acids will be incorporated into the various proteins that are sequestered in the cisternae of the rough endoplasmic reticulum. Dependent upon whether these proteins are collagen precursors, or proteins to be complexed with polysaccharide, they may follow at least two different routes through the cell, It is suggested that collagen precursors are secreted directly from the cisternae of the rough endoplasmic reticulum (small black arrow) either via direct, intermittent cisternal communications with the plasma membrane or via the formation of vesicles that eventually fuse with the plasma membrane and release their material. It is proposed that proteins to be complexed with polysaccharide are also sequestered in the cisternae of the rough endoplasmic reticulum, but that these proteins separate by vesicle formation from the rough endoplasmic reticulum in regions adjacent to the saccules and vesicles of the Golgi complex. These vesicles are presumed to merge with the Golgi cisternae where they may be complexed with substances such as polysaccharide and are subsequently secreted from the cell, again by the process of vesicle formation, migration, and fusion with the plasma membrane.

The Jibroblast and wound repair 67 unpublished observations). Chondroblasts also localize 35S-labelled sodium sulphate in the Golgi complex during chondroitin sulphate synthesis (Godman & Lane, 1964; Fewer et al. 1964). These observations and those of glu~ose-~H localization in the Golgi of intestinal cells (Peterson & Leblond, 1964; Neutra & Leblond, 1966) support the contention that this organelle is the site of polysaccharide synthesis, and may also be the region where polysaccharides are complexed to protein. Ross & Benditt (1964, 1965) noted zones in the fibroblasts that they interpreted as being actual or potential communications between the ergastoplasm and the extracellular spaces (PIS. 5, 8). They suggested that these may represent sites where collagen is secreted directly from the ergastoplasm to the extracellular space, as postulated in their hypothesis that collagen bypasses the Golgi zone. Thus at least two separate pathways for secretory protein may exist in the fibroblast (Text-fig. 2).

Chemical and autoradiographic studies have indicated both the similarities and possible differences between fibroblasts and other cells that synthesize protein for export, particularly concerning the role played by the Golgi in collagen synthesis. I t is probable that different pathways exist for different proteins synthesized and secreted by a given cell type and for different types of cells. The elucidation and specification of these pathways presents an interesting problem in understanding the function of each subcellular compartment.

Mucopolysaccharides in healing wounds Since the development of the concept of the ‘ground substance’ (Bensley, 1934),

a great deal of effort and speculation has been expended concerning the role of mucopolysaccharides in collagen formation and wound healing. The various components of the ‘ground substance’ are enumerated and discussed in numerous reviews (Gersh & Catchpole, 1949; Meyer, Hoffman & Linker, 1957; Dorfman, 1958; Muir, 1961 ; Fitton Jackson, 1964).

The presence of metachromatic material, specifically mucopolysaccharides, in healing wounds, is well known (Balazs & Holmgren, 1950; Berenson & Dalferes, 1960; Dunphy & Udupa, 1955; Kodicek & Loewi, 1955; Shetlar, Lacefield, White & Schilling, 1959; White, Shetlar & Schilling, 1961 ; White, Shetlar, Shurley & Schilling, 1965). As stated earlier, Dunphy & Udupa (1955) noted the appearance of hexosamine in granulation tissue and correlated this with the histochemical appearance of mucopolysaccharides. They found that the mucopolysaccharide content of wounds increased to a maximum between the third and sixth day after wounding, and thereafter decreased in amount. There appeared to be an inverse relation between the amount of mucopolysaccharide and the amount of collagen present in wounds. The increased amount of hexosamine within wounds at early time periods appears to be due to the presence of glycoproteins, derived largely from the serum. Boas (1953) had demonstrated that approximately 50 % of the hexosamine of rat connective tissues was due to extravascular serum proteins within the tissues. Therefore, the increased mucoprotein present in the early phases of wound healing appears to be due largely to serum mucoprotein, and is probably unrelated to the process of collagen synthesis (Jackson et al. 1960; Ahmad, 1961). This serum mucoprotein is clearly different from the meta-

5-2

68 RUSSELL Ross chromatic mucopolysaccharides formed during later periods in wounds (Guha et al. 1961).

The distribution of the various mucopolysaccharides within the ground substance has been reviewed by Meyer et al. (1957). Bentley (1967) has shown that less than 20% of the hexosamine of granulation tissue is derived from chondroitin sulphate and from traces of hyaluronic acid. The remainder is due to unidentified glycoproteins.

It is well established in 36S-sodium sulphate incorporation studies that fibroblasts can synthesize sulphated mucopolysaccharides both in healing wounds and in vitro (Gaines, 1960; Kennedy, 1960; Mancini, Vilar, Stein & Fiorini, 1961 ; Glucksmann, 1964). There is no evidence, however, that these substances play an active role in collagen formation. Furthermore, it was demonstrated by Gross (1957) that collagen fibrils can be formed in vitro without the participation of mucopolysaccharides.

Autoradiographic studies have demonstrated that the same cells that synthesize and secrete collagen are simultaneously synthesizing and secreting mucopolysaccharides. Stehbens (1962) has demonstrated that the fibroblasts, but not the endothelial cells, are responsible for mucopolysaccharide synthesis in healing wounds. Numerous investigators have suggested that the polysaccharides provide a stabilization mechanism for collagen molecules, fibres, and fibre networks. The inter-relationship between collagen and the mucopolysaccharides of the ground substance within healing wounds is not at all clear, and much work remains to be done before any role in stabilization of fibrils can be proven.

Chemical observations of healing wounds Woessner & Boucek (1961 a) examined numerous chemical parameters of healing

wounds in a series of studies of subcutaneously implanted polyvinyl sponges. Granula- tion tissue formed in polyvinyl sponges is similar both chemically and morphologically to that seen in healing wounds. They (Woessner & Boucek, 1961 a) observed a parallel increase in the DNA and ascorbic acid content of the granulation tissue up to the twentieth day. In addition, there was a positive correlation between increases in tensile strength and the hydroxyproline content of this tissue. Both of these reach a maximum as the connective tissues in the sponges mature (Woessner & Boucek, 1961 a ; Viljanto, 1964).

In examining the enzyme content of granulation tissue from sponges, Woessner & Boucek (1961b) noted several enzymes that increased in parallel with the DNA content. These included glutamic-oxalacetic transaminase, catalase, cytochrome C reductase, and phenolsulphatase. In addition, some lysosomal enzymes, notably a proteinase, acid phosphatase, and beta-glucuronidase also increased in parallel with DNA, and continued to increase after both DNA and collagen had reached a maximum. In contrast, other enzymes, notably alkaline phosphatase, peroxidase, and lactic and malic dehydrogenases increased in parallel with DNA, but decreased once the DNA content had reached a maximum. From these findings, these investigators concluded that of the three groups of enzymes, those that increased in parallel with DNA might be directly or indirectly related to collagen synthesis and the ones that

The jbroblast and wound repair 69 continued to increase after DNA had reached its maximum might be related to processes of collagen degradation and turnover since they have been identified as components of lysosomes. They presumed that enzymes in the last group, those that decreased after DNA had reached a maximum, were unreiated to collagen synthesis. Unfortunately, it is not clear which cells contain which enzymes at any given time. Because the fibroblast is a cell with many activities it is difficult to assign a functional significance to these enzymes in relation to collagen synthesis alone in the healing wound.

The reorganization or remodelling phase Cell turnover

Cell turnover in the intact dermis has been examined by looking for the presence of mitotic figures, after the administration of colchicine (Glucksmann, 1964), or by examining the utilization of tritiated thymidine (Grillo, 1963). In contrast to most adult tissues and organs (Chu, 1960; Bullough & Laurence, 1960a, b), wounds consistently demonstrate a high rate of cell turnover. In the intact ‘ hypodermis’, 7 % of all mitoses appeared to occur in fibroblasts, whereas migratory cells accounted for the bulk of the population that multiplied in this area (Chu, 1960). After the fibroblast population within a wound reached a maximum, cell turnover decreased to approximately the same level as that of the adjacent intact tissue.

Abercrombie (1964) has examined many aspects of the behaviour of cells toward one another in tissue culture. He has extensively studied the phenomena of contact inhibition and cell mobilization; two reactions intimately related to cell migration and mitosis. The factors that influence cell proliferation and migration in healing wounds are probably similar to those in tissue culture from which most of our conclusions have been derived.

Contact inhibition has been defined as ‘ . . .a failure. . .of one fibroblast to cross over the surface of another. . . Such inhibiting effects occur only on contact. . . ’ (Abercrombie & Heaysman, 1954). The loss of contact inhibition undoubtedly plays an important role in the migration of cells into a wound. The decrease in numbers of fibroblasts in older wounds may result from contact inhibition in a wound whose cell population has reached its maximum level.

Contact inhibition has also been shown to play a role in the synthesis of proteins in tissue culture. When normal human diploid fibroblasts, grown in culture, come into contact and become confluent, there is a decrease in the cellular uptake of amino acids, and of RNA and DNA precursors. These changes are associated with a decrease in free cytoplasmic ribosomes, and are reversible upon dissociating the cells (Levine, Becker, Boone & Eagle, 1965). Hence, contact inhibition appears to be important in the synthesis of new protein and in relation to cell division in vitro.

Cell mobilization, in contrast to contact inhibition, refers to the increased propensity of cells to move. This can be expressed as a given rate of cell migration. The nature of the stimuli that cause cells to divide and move is of obvious interest. Abercrombie proposes that diffusible substances, analogous to embryo extracts used in tissue culture to promote mitoses and cell movement (Willmer & Jacoby, 1936), may be

70 RUSSELL Ross responsible for these phenomena in a wound. Thus the old concept of a ‘wound hormone’ (Carrel, 1922) has been revived.

Other kinds of diffusible substances have also been suggested as being responsible for cell migration. Bullough (1962) suggested that each tissue may produce its own inhibitor which depresses the mitotic rate. By removing the tissues or blocking the inhibitor, the mitotic rate of cells would then increase. Bullough & Laurence ( 1 9 6 0 ~ ) observed that removal of skin from one side of a mouse ear resulted in increased mitosis on the other side. These observations led them to suggest that diffusible inhibitors from the operated side affected the intact tissues on the opposite side.

Turnover of extracellular substances The turnover of both cells and collagen in the intact adult dermis is relatively low

(Neuberger & Slack, 1953; Neuberger, 1955; Thompson & Ballou, 1956; Gerber, Gerber & Altman, 1960; Delaunay & Bazin, 1958), whereas the turnover of polysaccharides is quite high (Glucksmann, Howard & Pelc, 1956; Kennedy, 1960; Montagna & Hill, 1957; Glucksmann, 1964). Some adult tissues do display a rapid turnover of collagen. These include healing wounds (Boucek & Noble, 1961 ; Ross & Benditt, 1962b) and the involuting uterus (Harkness & Moralee, 1956). I n general, collagen turnover is more rapid in young growing animals (Harkness, Harkness & James, 1958; Lindstedt & Prockop, 1961) than in adult connective tissues.

The healing wound presents a picture of rapid turnover of cells, collagen, and mucopolysaccharides (Glucksmann, 1964; Lindsay & Birch, 1964). This rapid rate of turnover is maintained until such time as healing is complete, after which the metabolism of the scar approaches that of the previously intact connective tissue.

Collagenase studies The presence of a collagenase in vertebrate tissues has been conjectured for many

years, but no such enzyme had been identified until its discovery in the metamor- phosing tadpole tail (Gross & Lapiere, 1962; Gross, 1964). As an example of collagenolytic activity, this represents an interesting phenomenon, because large amounts of mature collagen are removed during the relatively short period of tail resorption. After the presence of a specific collagenase in the tadpole tail had been established (Gross & Lapiere, 1962; Gross, 1964), Eisen & Gross (1965) demonstrated that the collagenase in this particular system was synthesized by the epidermal cells of the tail. The enzyme appears to have a relatively short half life, is not stored in cells, but apparently is synthesized only under appropriate stimulation. A similar collagenase has recently been detected in healing wounds by Grillo & Gross (1967).

In contrast to the production of a collagenolytic enzyme by the epithelial cells, it has been demonstrated (Eisen & Gross, 1965) that the connective tissue cells of the resorbing tail are responsible for the production of a hyaluronidase. Therefore, it would appear that the dissolution of the various extracellular components of the connective tissues of the resorbing tadpole tail are controlled in part by epithelium and in part by the connective tissue cells.

An interesting corollary to the collagenase system in the tadpole is that described

The fibroblast and wound repair 71 by Wassermann (1956). He observed resorption of degenerated material in the sub- epithelial space around periodontal fibres in the guinea-pig and suggested that a proteolytic enzyme was produced by the epithelium.

During the early phases of wounding, collagen fibres are degraded in the presence of the many inflammatory cells. It has been established that granules of these cells contain acid hydrolytic enzymes (lysosomal enzymes, such as cathepsins) (Hirsch, 1965). These enzymes probably play a role in the dissolution of the already altered collagen in the initial wound site. However, as noted previously, collagen turnover appears to be reasonably rapid during the later phases of healing. This is probably due in part to the greater solubility characteristics of the newly formed collagen molecules (Gross, et aE. 1955; Jackson & Fessler, 1955; Gross, 1958). Jackson (1958) and Jackson & Bentley (1960) have pointed out that the most recently formed collagen in the healing wound is the most soluble. However, it is probably true that a certain amount of old collagen is also removed. Both collagenolytic and proteolytic activity are present in wounds although no definitive information is yet available concerning the source of the collagenase.

111. WOUND CONTRACTION

Numerous explanations have been given for the process of wound contraction that results in a marked decrease in wound size. Among the factors that have been implicated are changes in the dimensions of collagen fibres in the surrounding skin, movement of all of the cells within the granulation tissue (Abercrombie, James & Newcombe, 1960), or of a specialized zone of cells located at the margin between the wound and the intact tissue. The pull of the contracting repair tissue has been shown to be sufficient to cause intussusception of an island of intact tissue, when this tissue was left in the middle of the wound (Billingham & Medawar, 1955). In a similar histologic study of wound contraction, Luccioli, Kahn & Robertson (1964) demonstrated that the corium of the wound margins could ‘slide’ over the granulation tissue, and in hori- zontal sections the new collagen fibres displayed a circumferential arrangement around the centre of the wound.

Recent evidence indicates that the contractional forces arise from the cells within the granulation tissue. Wound contraction occurred to the same extent in normal and scorbutic wounds. Scorbutic wounds are highly cellular yet contain little or no collagen (Abercrombie, Flint & James, 1956; Grillo & Gross, 1959). Additional evidence to indicate that contraction is cell mediated was given by James (1964) who observed that potassium cyanide can inhibit the contractional force. He went on to demonstrate that neither change in pH nor the presence of potassium ions influenced wound contraction.

As a wound contracts there is an associated increase in the amount of both collagen and non collagenous protein, fluid and per unit area thickness of the wound (Luciolli et al. 1964).

Perhaps the most important conclusion that can be drawn from these investigations is the fact that wound contraction appears to be dependent upon the various co- ordinated activities of the cells within the wound. Wound closure is effected by the

72 RUSSELL Ross cells by synthesizing collagen, and by a centripetal pull on the surrounding tissues so that the size of the wound is reduced, and that the edges are united sooner than they would have been had contraction not taken place.

IV. FACTORS AFFECTING WOUND REPAIR

Nutritio?Z It is often difficult to correlate the results of different experiments concerning the

effects of systemic factors upon wound healing because of different experimental conditions. This review will not attempt to be complete, but rather will point to areas of interest where more information is necessary if we are to understand the effects of these various agents.

Dietary elements known to have the most profound effect upon connective tissue formation include proteins, amino acids, and ascorbic acid. These are often inter- related. An understanding of their action provides further insight into the cellular mechanisms involved in the formation of connective tissue.

Protein deficiency Prolonged protein starvation has a deleterious effect upon wound healing in

experimental animals (Howes, Briggs, Shea & Harvey, 1933 ; Levenson, Burkhill & Waterman, 1950; Stahl, 1962, 1963). Kobak, Benditt, Wissler & Steffee (1947) observed a marked decrease in tensile strength in wounds in protein-deficient animals. The decreased tensile strength observed in this study corresponded with a decrease in both the number of fibroblasts and the amount of collagen formed within the wounds.

I t was further observed that the administration of methionine to protein-starved rats had a beneficial effect upon connective tissue formation (Localio, Morgan & Hinton, 1948). This effect upon wound regeneration has been confirmed several times, although the role played by this amino acid remains unclear (Williamson, McCarthy & Fromm, 1951; Perez-Tamayo & Ihnen, 1953; Udupa, Woessner & Dunphy, 1956). Williamson & Fromm (1953) found that in protein starvation cystine had the same beneficial effect as methionine on wounds, a finding subsequently confirmed by Fromm & Nordlie (1957). Collagen contains less than I % methionine and no cystine, yet cystine was the limiting amino acid upon wound repair (Williamson, 1956; Williamson & Fromm, 1955). In healing wounds methionine appeared to be the primary source of cystine yet cystine did not appear in collagen or in the form of sulphated polysaccharides (Williamson, Gagan, Haley & Williamson, 1965). These studies indicate a supporting role for the sulphated amino acids in the various cellular mechanisms involved in synthesis and secretion by connective tissue cells. Much of the earlier work in this field has been summarized by Williamson (1957). Unfortunately, there have been no fine structure studies of the cells in these circumstances.

The jibroblast and wound repair 73

Ascorbic acid Defective wound healing in ascorbic acid deficiency (scurvy) has been recognized for

centuries. In the eighteenth century (Anson, 1748; Lind, 1772) it was noted that individuals whose diet was lacking in fresh fruits and vegetables were prone to develop scurvy. At the same time it was observed that healed scars would rupture in persons afflicted with this disease. The necessity for ascorbic acid, not only in collagen formation, but also for its maintenance in the healed wound is now well established. Histo- logic and biochemical evidence correlating ascorbic acid concentration in wounds with collagen synthesis has been presented by numerous investigators (Bourne, I 946 ; Pirani & Levenson, 1953; Abt, Von Schuching & Roe, 1959~2, by 1960; Abt & Von Schuching, 1961 ; Chen & Postlethwait, 1961).

In 1942, Wolbach & Bessey observed that collagen was either not produced or was produced in defective form in scurvy. These and other investigators noted ‘vacuolation ’ in scorbutic wound fibroblasts that were prepared for routine histologic examination (Penney & Balfour, 1949; Person, 1953). They presumed that the vacuoles contained the source of the extracellular liquids described in scorbutic animals by Wolbach and postulated that these liquids might represent a collagen precursor stored within the cells. Fine structure observations of wounds suggest that these vacuoles represent both large deposits of lipid and possibly the large, dilated ergastoplasmic cisternae so common in scorbutic fibroblasts (Ross & Benditt, 1962a, 1964; Peach, 1962).

Wolbach & Howe (1926) and Wolbach (1933) described a substance in the extracellular spaces of bone and dentine that lacked the ordinary staining characteristics of collagen. Following the administration of ascorbic acid to scorbutic animals, argyro- philic fibres were observed in the extracellular matrix within 24 hr.

Effects of scurvy upon the jibroblast Numerous subcellular alterations have been described within the scorbutic fibro-

blast in healing wounds by Ross & Benditt (1962a, b, 1964), Peach (1962)~ and Jorgensen (1964 b). The changes delineated by these investigators include :

(I) Two effects on the ergastoplasm of the fibroblast including ( a ) a rounding up or ‘vacuolation’ of the cisternae (Pls. 12, 14); (b) a loss of the characteristic configuration of the aggregates of ribosomes attached to the surface of the membranes of the rough endoplasmic reticulum in the fibroblast (PIS. 12-14). In normal cells these aggregates take the form of curved profiles and often occur in paired rows (Pls. 4,6). In scorbutic fibroblasts the ribosomes appear to be randomly distributed upon the surface of the ergastoplasmic membranes, in contrast to those seen in neighbouring macrophages in the scorbutic wound (PI. 13). This is not true for all scorbutic fibroblasts; however, a general loss or diminution of the characteristic pattern can be observed.

(2) An increase in the number of free cytoplasmic ribosomes. (3) The appearance of large numbers of lipid deposits within the cytoplasm. (4) A lack of collagen in the extracellular spaces, although individual fibrils can

occasionally be seen. Instead, the bulk of the extracellular regions contain large mats

74 RUSSELL Ross of fine filamentous material, some of which appears to be fibrin, although much of this material is difficult to identify (Pl. 14) .

In addition, Ross & Benditt ( 1 9 6 2 ~ ) have described large numbers of phagocytic cells containing ingested erythrocytes and large amounts of ferritin within phagocytic vacuoles in scorbutic wounds (Pl. 13). This presumably resulted from the increased haemorrhage commonly seen in scurvy.

A study of the fine structure of scorbutic wounds before and after the administration of ascorbic acid (Ross & Benditt, 1964) demonstrated similar alterations in fine structure in the scorbutic fibroblast. Within 4 hr. after the administration of ascorbic acid, the reappearance of ribosomal aggregates attached to the membranes of the cisternae of the ergastoplasm, characteristic of non-deficient fibroblasts, was noted (Pl. I S ) . Within 12 h., extracellular collagen fibrils appeared and larger numbers of aggregates of attached ribosomes were present (PI. I S ) . After 24 hr., the previously scorbutic fibroblasts were indistinguishable from normal cells and large amounts of collagen were present in the extracellular spaces.

It is important to point out that the specificity of the morphologic alterations of the cells during scurvy remains unclear. Similar changes in fine structure occur in cells following injury resulting from various toxic agents. I t is possible that the alterations in the scorbutic fibroblast represent a non-specific response to injury, or a non-specific interference with the protein synthetic apparatus resulting in its disassembly. As yet we have little understanding of the factors controlling the pattern of ribosomes and their attachment to the membranes of the rough endoplasmic reticulum, both obviously related to the changes seen in scurvy and other defects in cells synthesizing protein.

Extracellu~ar substances in scorbutic wounds In a series of autoradiographic investigations utilizing tritiated proline, it was noted

(Ross & Benditt, 1962b) that scorbutic fibroblasts are capable of taking up labelled proline and releasing it into the extracellular spaces. I t is possible that part of the extracellular label present in scorbutic wounds represents plasma proteins, including fibrin, from the wound exudate. Increased capillary fragility is a well known phenomenon in scurvy and it is conceivable that much labelled plasma protein passes into the wound. It is also possible that scorbutic cells may be synthesizing an abnormal protein. The nature of the extracellular material in scorbutic wounds has remained unclear.

There is evidence to indicate that the material that accumulates in the extracellular spaces during scurvy is not a collagen precursor. In 1953, Robertson & Schwartz studied the carrageenin granuloma in ascorbic acid deficient animals. After the administration of ascorbic acid, they observed a more rapid formation of collagen during the recovery than occurred in normal animals. Robertson et al. (1959) also studied this problem with labelled amino acids. They gave proline to normal and scorbutic animals that subsequently received ascorbic acid, and then isolated proline and hydroxyproline from each of the granulomas. Most of the collagen in these studies was synthesized during the recovery phase, and did not appear to arise from a pre- formed, proline-rich precursor. Gould & Woessner (1957) made similar observations in healing skin wounds from normal and scorbutic guinea-pigs. Gould, Manner,

The jibroblast and wound repair 75 Goldman & Stolman (1960) were also unsuccessful in isolating a proline-rich precursor from granulation tissue that could have served as a reservoir for the formation of hydroxyproline in collagen.

Ross & Benditt (1964) examined the susceptibility of the extracellular material in scorbutic wounds to both collagenase and trypsin. They found that the material was removed by trypsin and unaffected by collagenase, again supporting the contention that this material does not represent a collagen precursor. These observations, combined with the changes in fine structure observed after ascorbic acid administration, support the hypothesis that intracellular collagen synthesis occurs at an extremely rapid rate following the administration of ascorbic acid to scorbutic animals. It does not appear that there is any conversion of an extracellular precursor to collagen in the presence of ascorbic acid.

A decrease in the neutral salt soluble fraction of dermal collagen in scorbutic guinea-pigs was observed by Gross (1958, 1959). As a result of these and other observations, he felt that in scurvy a deficiency in collagen formation occurred prior to the stage of fibril aggregation. He stated that ‘a deficiency of ascorbic acid either interferes with the synthesis of new collagen in intact skin or causes its destruction and removal ,as rapidly as it is produced ’. The cellular alterations previously described would support the notion that synthesis is interfered with in scurvy. The possible chemical sites of action of ascorbic acid are generally thought to be located at either of two points in the synthetic pathway. It has been suggested that either (a) in scurvy a collagen precursor is synthesized containing peptide-bound proline, approximately half of which becomes hydroxylated after the administration of ascorbic acid (Stetten, 1949; Robertson, 1952; Gould & Woessner, 1957) or (b) that hydroxyproline in collagen was derived from proline and hydroxylated before incorporation into collagen, or a peptide precursor of collagen (Stone & Meister, 1962; Schauble, Chen, Postle- thwait & Dillon, 1960; Mitoma & Smith, 1960).

As was mentioned previously, a major part of collagen synthesis was found in the microsomal fraction obtained from fibrogenic cells (Lowther et al. 1961 ; Prockop, Peterkofsky & Udenfriend, 1962). Autoradiographic techniques demonstrate that synthesis appears to occur on the ribosomes of the rough endoplasmic reticulum of the connective tissue cell (Ross & Benditt, 1965; Revel & Hay, 1963).

Peterkofsky & Udenfriend (1961, 1963) studied cell-free systems derived from chick embryos and were able to demonstrate an incorporation of proline into peptide and a conversion to peptide-bound hydroxyproline in these systems. Lukens (1965) also examined chick embryo minces and found that the substrate for the hydroxylation reaction was proline in a polypeptide rather than a species of prolyl-s-RNA. He was unable to detect synthesis of hydroxyprolyl-s-RNA under conditions when prolyl-s- RNA was synthesized from appropriate chick embryo extracts. In addition, puromycin inhibited the incorporation into protein of proline and hydroxyproline to an equal extent (Juva & Prockop, 1964; Lukens, 1965).

In contrast, Manner & Gould (1962, 1963) found an ‘active’ hydroxyproline in the form of an s-RNA hydroxyproline complex indicating that some hydroxylation may occur prior to peptide synthesis. These conflicting results remain to be settled. The

76 RUSSELL Ross problem has been reviewed in detail by Gould (1963). The results of the various chemical, autoradiographic and morphologic studies point to the fact that both hydroxylation of proline and its incorporation into protein occur within the cell.

It is conceivable that the rounded and dilated cisternae of rough endoplasmic reticulum seen in scorbutic fibroblasts may contain large peptides or proteins, rich in proline, and lacking in hydroxyproline, thus explaining the relatively rapid reversion to normalcy following ascorbic acid administration.

Ascorbic acid appears to have a direct local effect upon collagen formation. Gould (1958) studied polyvinyl sponges implanted bilaterally in the skin of scorbutic guinea- pigs. Ascorbic acid, in an amount insufficient to have a systemic effect, was added to a sponge on only one side of each animal. Rapid synthesis of collagen was observed in the vitamin C-treated sponge, whereas none was found in the untreated sponge from the other side of the same animal. Further experiments indicating a local action of this vitamin upon collagen synthesis have been performed by several workers (Abt, Von Schuching & Roe, 1960; Postlethwait, Chen, Kamin & Dillon, 1960; Schauble et al. 1960). These investigators demonstrated an accumulation of ascorbic acid at sites of active synthesis such as healing wounds.

Robertson & Hewitt (1961) were able to demonstrate an effect of ascorbic acid in vitro on suspensions of scorbutic granuloma cells. In these studies they found a similar enhancement of collagen formation upon adding ascorbic acid to the suspension medium compared to that seen after the intraperitoneal administration to scorbutic animals.

No definitive information is available concerning the turnover of collagen in old scar tissue as compared with uninjured tissues of the same type. Old scars have been said to rupture during scurvy. Studies of the turnover of collagen in scars would undoubtedly further our understanding of the role of ascorbic acid in the maintenance of scar tissue.

Mucopolysaccharide formation in scurvy Vitamin C also has a salutary effect upon mucopolysaccharide metabolism (Brad-

field & Kodicek, 1951; Bowness, 1957). Dunphy, Udupa & Edwards (1956) studied healing wounds in normal and scorbutic guinea-pigs before and after the administration of ascorbic acid. They found a defect in mucopolysaccharide formation in scurvy. However, they also noted an inverse relationship between collagen formation and the presence of hexosamine, determined chemically, after giving ascorbic acid to the animals. These findings confirmed similar studies in which a decreased uptake of 35S-sodium sulphate in scorbutic granulation tissue was noted with no change in the amount of hexosamine containing the labelled isotope (Kodicek & Loewi, 1955; Upton & Odell, 1956). This decreased incorporation of inorganic sulphates was thought to be due to a defect in sulphation of mucopolysaccharides during scurvy. This interpretation was supported by Robertson & Hinds (1956) and Slack (1958) who demonstrated that hyaluronic acid accumulated in scorbutic wounds but that chondroitin sulphate formation did not occur. As yet, no relationship between the lack of formation of sulphated mucopolysaccharide within scorbutic wounds, and the

The jibroblast and wound repair 77 lack of collagen synthesis has been demonstrated. At one time mucopolysaccharides were considered to be essential for the formation of collagen fibrils; however, Gross & Kirk (1958) were able to demonstrate that mucopolysaccharides have no effect on the rate or extent of fibril formation in vitro. Ross & Benditt (1965) have suggested that mucoproteins and collagen are synthesized and secreted via separate pathways in the fibroblast. It would appear that these two substances play an integral and undoubtedly important structural role in the connective tissue ; however, any direct relationship between mucopolysaccharides and collagen molecules in the formation of collagen fibrils remain to be discovered.

Steroid Hormones Studies of the effects of adrenal steroids upon wound healing are both conflicting

and confusing. I t is particularly difficult to evaluate the significance of many of the investigations with these agents, as it is well known that different animals differ in their susceptibility to steroids. In addition, problems of inanition, particularly in the use of cortisone, have been demonstrated with relatively low doses of this hormone. Therefore, these factors must be taken into account when evaluating the effects of various drugs (Di Pasquale & Steinetz, 1964).

Two effects of steroids have been demonstrated upon connective tissues. These are the well-known anti-inflammatory action, and an apparently direct effect upon the connective tissue forming cells in general.

Both cortisone and hydrocortisone have been shown to inhibit the synthesis of connective tissues in vitro and in vivo (Ragan et al. 1949; Porter, 1951 ; Chassin et al. 1954; Taubenhaus, 1953 ; Taubenhaus, Jacobson, Morton & Levine, 1953). Billing- ham & Russell (1956) also demonstrated an inhibition of granulation tissue formation following the administration of cortisone. Similarly, a decreased uptake of glycineJ4C into the organic matrix of bone, when collagen-forming bone fragments were studied in vitro, was observed by Vaes & Nichols (1962).

Nocenti, Lederman, Furey & Lopano (1964) studied the effects of cortisone and hydrocortisone upon the connective tissue within subcutaneously implanted polyvinyl sponges. They noted that there was a decrease in both the wet and the dry weights of the granulation tissue, a decrease in the amount of protein-bound hydroxyproline, and a general decrease in total collagen formation, particularly in the salt-soluble fraction of the collagen extracted from the sponges from the treated animals. They interpreted their results as indicating that steroids may influence protein synthesis, but not specifically collagen synthesis.

Several investigators (Bangham, 1951 ; Jorgensen, 1963 a) suggested that the primary effect of cortisone and hydrocortisone upon cutaneous wounds resulted from the suppression of the initial inflammatory response by these steroids. A suppression of granulation tissue formation in turpentine abscesses in mice was observed if cortisone was administered 1-3 days before injuring the animals (Spain et al. 1952). If the steroid was given 2 days after injury, healing in the treated animals could not be distinguished from the controls. Savlov & Dunphy (1954) demonstrated similar results by measuring the tensile strength of wounds in rats treated with cortisone. A reduc-

78 RUSSELL Ross tion in tensile strength of corneal wounds was also observed after the administration of prednisolone (Aquavella, Gasset & Dohlman, 1964). If the animals were wounded and the wounds allowed to heal for 10 days before the steroid was given, healing did not appear to be retarded. A similar retardation in pre-treated wounds was observed by Sandberg (1964c), whereas if he waited for 2 days before giving the steroid, again no effect was noted. The latter studies suggest that the primary effect of steroids is upon the inflammatory response rather than upon collagen synthesis. They do not explain, however, the anabolic effect demonstrated in vitro.

Jorgensen (1964~) examined the fine structure of wounds in rats pretreated with prednisone for five days prior to wounding. He felt that the fibroblasts seemed ‘less mature’ in the steroid-treated wounds. This is a difficult judgement to make and, unfortunately, there have been no systematic studies of the effects of steroids upon the fine structure of the various connective tissue cells.

Examination of the effect of cortisol upon other intercellular substances in connective tissue in the cotton ball granuloma revealed a decrease in the acid mucopolysaccharide content of these granulomas (Likar et ul. 1963). Schiller & Dorfman (1957) observed a decreased uptake of 14C-acetate by both hyaluronic acid and chondroitin sulphate, and of s6S-sulphate by chondroitin sulphate, in the skin of rats pretreated with cortisone acetate. Similarly, Schiller, Blumenkrantz & Dorfman (1965) found a decrease in the concentration of both chondroitin sulphate and hyaluronic acid in skin following treatment with hydrocortisone, whereas they noted a decrease in chondroitin sulphate but an increased hyaluronic acid content after prednisolone treatment. Both of these steroids exert an anti-inflammatory effect, yet each appears to have a selective action upon these two components of the connective tissues.

An interesting suggestion was made by Sandberg & Steinhardt (1964). They felt that cortisone may produce a decrease in the enzyme histidine decarboxylase, and thereby interfere with the local release of histamine in the wounds. A discussion of histamine can be found below. It has been stated in a number of studies that histamine accelerates the formation of collagen in wounds and thereby increases the tensile strength of these wounds.

I t can be seen from the preceding discussion that adrenal corticosteroids may have multiple effects, including an anti-inflammatory response and a depression of protein and polysaccharide synthesis. These responses appear to be dose related (Kivirikko, 1963). Their significance awaits further investigation into the role of steroids as they affect the dynamic equilibrium present between the various extracellular substances and the cells.

Kelly (19624 b) has studied the effect of oestrogen upon connective tissues grown in subcutaneously implanted polyvinyl sponges. He noted that there appeared to be a prolongation of tissue growth within oestrogen-treated polyvinyl sponges, in contrast to untreated controls; and suggested that the increase in the cell numbers and vas- cularity of the sponges was related to oestrogen treatment.

A decrease in tensile strength was observed in uterine wounds during pregnancy (Hooker, 1941). Morgan (1963) and Kao, Hitt, Bush & McGavack (1964) have indicated that oestradiol caused an increase in collagen synthesis within the uterus ; however,

The jibroblast and wound repair 79 a concomitant increase in catabolism produced some decrease in collagen concentration in this organ (Kao, Hitt & McGavack, 1965). The effects of this hormone upon collagen synthesis and degradation in the uterus seem reasonably clear, although the means by which they are exerted are poorly understood (Morgan, 1963; Smith & Kaltreider, 1963 ; Fainstat, 1962). The specific effects and inter-relationships of oestrogen with the synthesis of collagen in wounds remain to be determined.

Histamine Mast cells, known to contain relatively large quantities of histamine (Benditt &

Lagunoff, 1964)~ decrease in the neighbourhood of a wound within the first 24 h., and return to normal numbers within the next 8 days (Wichmann, 1955; Miller & Whitting, 1965). In addition, wounds in rat skin contain large amounts of histidine decarboxylase, resulting in histamine release (Kahlson et al. 1960). This has been correlated with an increase in wound tensile strength. It has been suggested that histamine plays an important role via mast cells in stimulating the fibroplasia that ensues as a result of wounding (Kelsall, 1961 ; Miller & Whitting, 1965).

Sandberg (1964b) noted that inhibition of histamine formation caused a decrease in collagen synthesis in granulation tissue. He studied rats on a pyridoxine-deficient diet that was supplemented with semicarbazide. Such a diet tends to inhibit the formation of histamine. Wounds from animals on this diet contained less hydroxyproline or collagen, than controls. A decreased tensile strength of wounds from the treated animals was also observed. Upon return of the animal to a normal diet, wound tensile strength increased. Boyd & Smith (1959) noted that the administration of histamine liberators, such as 48-80, had the effect of increasing wound tensile strength. Similar observations were made by Sandberg (1964~) on granulation tissue in rats in which he related the histamine forming capacity of the animal directly to its ability to synthesize collagen.

V. SUMMARY

This review of connective tissue repair has attempted to place into historical perspective information obtained by newer approaches. The literature review is incom- plete, as it was unfortunately necessary to leave many interesting studies out of the discussion. Emphasis has been placed upon what is known of the inflammatory response, the fine structure of the connective tissue cells in healing wounds and with correlated chemical findings in these tissues.

An optimal inflammatory response appears to be an important, rapid, non-specific stimulus for fibroplasia. It is not clear how inflammation exerts this effect. The inflammatory cells and their enzymes markedly alter the extracellular matrix of injured tissue. The matrix of connective tissue may itself participate in the control of its own synthesis and degradation. It is possible that modification of this environment by injury and/or inflammation with ensuing matrix alteration may provide a stimulus for cell migration and protein synthesis. The converse may also be true, that is, a given level of matrix concentration may have an inhibitory effect upon the connective tissue cells. The inter-relationships between the connective tissue matrix and the cells, and the

80 RUSSELL Ross possibilities of feedback mechanisms playing a role in maintaining a balance between these two are important areas for future investigation. In this regard, additional questions may be asked concerning the role of the fibroblast in remodelling and degradation of connective tissue. It is not yet clear how important a balance between collagenolytic enzymes and the solubility states, or stability, of collagen are in each connective tissue. It will be interesting to determine which cells make collagenolytic and/or proteolytic enzymes upon appropriate stimulus.

It is possible to distinguish between the fibroblast and the monocyte, or potential macrophage with the electron microscope. The rough endoplasmic reticulum with its large numbers of attached ribosomes is extensively developed in the fibroblast in contrast to the monocyte. The endoplasmic reticulum sequesters collagen precursors and other secretory proteins for transport either directly to the extracellular space, as appears to be the case for collagen, or to the Golgi complex as is the case for other ex- portable proteins. Collagen precursors are secreted into the environment and are not shed from within the cell surface.

A number of cytoplasmic alterations have been described for fibroblasts and other cells during various pathological states. The significance of these alterations is not clear. It will be important to distinguish between specific and non-specific responses to injury, if these alterations are to aid us in understanding the various cellular responses.

The source of the fibroblasts in granulation tissue appears to be mesenchymal cells from adjacent tissues rather than blood-borne precursors. Although contact inhibition can be demonstrated in vitro, it is not clear how important this phenomenon is in vivo, nor are the reasons for the ability of some tissues to heal by regeneration rather than by scar tissue formation understood.

These and many other questions remain to be answered. The healing wound is multifaceted and presents the opportunity for systematic investigation into the problems of cell proliferation, cell and matrix interactions, and protein synthesis in vivo and it also can help to further our understanding of the ubiquitous fibroblast and its complex extracellular matrix.

The author is deeply indebted to several friends and colleagues who aided in the preparation of this review by numerous discussions. Particular thanks are due George Odland for his critical commentary of the manuscript and to Audrey Glauert and John Dingle for their thought-provoking ideas and suggestions.

I am most grateful to Professor Earl P. Benditt for his constant support and enthusiasm over the years. Working with him has served as a stimulus for new thoughts and ideas, and has provided many hours of pleasurable discussion.

Mrs Dorris Knibb deserves particular thanks for her assistance in typing and editing the manuscript, and Mr Johsel Namkung for preparing the photographic prints.

The work done in the laboratories has been supported by grants DE-01703 and HE-3174 from the U.S. Public Health Service. Dr Ross is the holder of a Career Development Research Award from the US. Public Health Service, No. DE-09503.

The jibroblast and wound repair 81

VI. REFERENCES

ABERCROMBIE, M. (1964). Advances in biology of skin. Vol. v. Wound Healing pp. 95-112. Eds. W. Montagna and R. E. Billingham. London.

ABERCROMBIE, M., FLINT, M. H. & JAMES, D. W. (1956). Wound contraction in relation to collagen formation in scorbutic guinea pigs. J . Embryol. exp. Morph. 4, 16775.

~ E R C R O M B I E , M. & HEAYSMAN, J. E. H. (1954). Observations on the social behaviour of cells in tissue culture. 11. ‘Monolayering’ of fibroblasts. Expl. Cell Res. 6, 293-306.

ABERCROMBIE, M., JAMES, D. W. & NEWCOMBE, J. F. (1960). Wound contraction in rabbit skin, studied by splinting the wound margins. J. Anat. 94, 170-82.

ABT, A. F. & VON SCHUCHING, S. (1961). Catabolism of ~-ascorbic-~-Cl~ acid as a measure of its utilization in the intact and wounded guinea pig on scorbutic maintenance and saturation diets. Ann. N. Y. Acad. Sci. 92, 148-58.

ABT, A. F., VON SCHUCHING, S. & ROE, J. H. (1959a). Connective tissue studies. I. Relation of dietary and tissue levels of ascorbic acid to the healing of surgically-induced wounds in guinea pigs. Johns Hopkins Hosp. Bull. 104, 16373.

ABT, A. F., VON SCHUCHING, S. & ROE, J. H. (19596). Connective tissue studies. 11. The effect of vitamin C deficiency on healed wounds. Johns Hopkins Hosp. Bull. 105, 67-76.

ABT. A. F. VON SCHUCHING, S. & ROE, J. H. (1960). Connective tissue studies. 111. Ascorbic acid, collagen and hexosamine distribution and histology of connective tissue in scars produced in guinea pigs on various vitamin C dietary levels following wounding by abdominal incision. J. Nutr. 70, 427-37.

AHMAD, M. (1961). Glucosamine and hydroxyproline content of granulation tissue on different days of wound healing in white albino rats. Ann. Biochem. exp. Med. 21, 295306.

ALLGOWER, M. (1956). The cellular basis of wound repair. Springfield, Ill. ALLGOWER, M. & HULLIGER, L. (1960). Origin of fibroblasts from mononuclear blood cells: A study on

in vitro formation of the collagen precursor, hydroxyproline, in buffy coat cultures. Surgery 47, 603-10.

ANSON, G. (1748). A voyage around the world (compiled by Richard Walter). London: John and Paul Knopton.

AQUAVELLA, J. V., GASSET, A. R. & DOHLMAN, C. H. (1964). Corticosteroids in corneal wound healing. Am. J. Ophthal. 58,621-6.

AREY, L. B. (1936). Wound healing. Physiol. Rev. 16, 327-406. BALAZS, A. & HOLMGREN, H. J. (1950). The basic dye-uptake and the presence of a growth-inhibiting

BALOGH, K., JR. & HAJEK, J. V. (1965). Oxidative enzymes of intermediary metabolism in healing bone

BANGHAM, A. D. (1951). The effect of cortisone on wound healing. Br. J . exp. Path. 32, 77-84. BARNHART, M. I. (1965). Importance of neutrophilic leukocytes in the resolution of fibrin. Fedn. Proc.

BEAR, R. S. (1952). Advances in protein chemistry, Vol. VII, pp. 69-160. Eds. M. L. Anson, K. Bailey and

BENDITT, E. P. & LAGUNOFF, D. (1964). The mast cell: its structure and function. Prog. Allergy 8,

BENSLEY, S. H. (1934). On the presence, properties, and distribution of the intercellular ground sub-

BENTL~Y, J. P. (1967). Rate of chondroitin sulphate formation in wound healing. Ann. Surg. 165,186-91. BERENSON, G. S. & DALFERES, E. R. (1960). Identification of acid mucopolysaccharides from granulation

BILLINGHAM, R. E. & MEDAWAR, P. B. (1955). Contracture and intussusceptive growth in the healing

BILLINGHAM, R. E. & RUSSELL, P. S. (1956). Studies on wound healing, with special reference to the

BLOOM, W. (1927). Transformation of lymphocytes of thoracic duct into polyblasts (macrophages) in

BOAS, N. F. (1953). Method for the determination of hexosamines in tissues.J. biol. Chem. 204, 553-64. BORNSTEIN, P., KANG, A. H. & PIEZ, K. A. (1966). The nature and location of intramolecular cross-

BOUCEK, R. J. & NOBLE, N. L. (1961). Metabolism of collagen. Appearance and disappearance of (14C)

BOURNE, G. H. (1946). The effect of vitamin C on the healing of wounds. Proc. Nutr. SOL. 4, 204-10.

substance in the healing tissues of skin wounds. Expl. Cell Res. I, 206-16.

fractures. A histochemical study. Am. J. Anat. 116, 429-48.

Fedn Am. Socs. exp. Biol. ~ q , 846-53.

J. T. Edsall. New York.

195-223.

stance of loose connective tissue. Anat: Rec. 60, 93-110.

tissue in rats. Br. J . exp. Path. 41, 422-9.

of extensive wounds in mammalian skin. J. Anat. 89, I 14-23.

phenomenon of contracture in experimental wounds in rabbits’ skin. Ann. Surg. 14, 961-81.

tissue culture. Proc. SOC. exp. Biol. Med. 24, 567-9.

links in collagen. Proc. natn. Acad. Sci., U . S . A . 55, 417-24.

hydroxylysine in rat connective tissue. Biochem. J . 80, 148-54.

6 Biol. Rev. 43

82 RUSSELL Ross Bow~s, J. H. & KENTEN, R. H. (1948). The amino-acid composition and titration curve of collagen.

BOWNESS, J. M. (1957). The effect of ascorbic-acid deficiency on the concentration of acid mucopoly-

BOYD, J. F. & SMITH, A. N. (1959). The effect of histamine and a histamine-releasing agent (compound

BRADFIELD, J. R. G. & KODICEK. E. (19 jr). Abnormal mucopolysaccharide and ‘precollagen’ in vitamin

BUGLIARI, G. R. (1927). Influenza dei trefoni embrionari e leucocitari sulla guariglone delle ferite da

BULLOUGH, W. S. (1962). The control of mitotic activity in adult mammalian tissues. Biol. Rev. 37,

BULLOUGH, W. S. & LAURENCE, E. B. (1960a). The control of epidermal mitotic activity in the mouse.

BULLOUGH, W. S. & LAURENCE, W. B. (1960b). The control of mitotic activity in mouse skin. Dermis

BUNTING, H. &WHITE, R. F. (1950). Histochemical studies of skin wounds in normal and in scorbutic

CAMERON, D. A. (1961). The fine structure of osteoblasts in the metaphysis of the tibia of the young

CAMERON, G. R. (1951). Pathology of the cell. Springfield, Ill. CARO, L. G. & PALADE, G. E. (1964). Protein synthesis, storage, and discharge in the pancreatic exocrine

cell. An autoradiographic study. J. Cell Biol. 20, 473-95. CARREL, A. (1921). Cicatrization of wounds. XII. Factors initiating regeneration. J. exp. Med. 34,

425-34. CARREL, A. (1922). Growth-promoting function of leucocytes. j . exp. Med. 36, 385-92. CARREL, A. (1930). Process of wound healing. Proc. Inst. Med., Chicago 8, 62-6. CARREL, A. & EBELING, A. H. (1926). The transformation of monocytes into fibroblasts through the

action of Rous virus. J . exp. Med. 43, 461-8. CHALKLEY, H. W., ALGIRE, G. H. &MORRIS, H.P. (1946). Effect of the level of dietary protein on vascular

repair in wounds. J. natn. Cancer b s t . 6, 363-72. CHAPMAN, J. A. (1961). Morphological and chemical studies of collagen formation. I. The fine structure

of guinea-pig granulomata. J. biophys. biochem. Cytol. 9, 639-51. CHASSIN, J. L., MCDOUGALL, H. A., STAHL, W., MACKAY, M. & LOCALIO, S. A. (1954). Effect of

adrenalectomy on wound healing in normal and in stressed rats. Proc. SOC. exp. Biol. Med. 86, 446-8. CHEN, R. W. & POSTLETHWAIT, R. W. (1961). Ascorbic acid in the biosynthesis and maintenance of

collagen. Surgery Gynec. Obstet. 112, 66774. CHEN, R. W. & POSTLETHWAIT, R. W. (1964). The biochemistry of wound healing. Mongr. Surg. Sci. I,

215-76. CHU, C. H. U. (1960). A study of the subcutaneous connective tissue of the mouse, with special reference

to nuclear type, nuclear division and mitotic rhythm. Anat. Rec. 138, 11-26. CLIFF, W. J. (1963). Observations on healing tissue: a combined light and electron microscopic investi-

gation. Phil. Trans. R. Sac. B 246, 305-26. COHN, Z. A. & HIRSCH, J. G. ( 1960). The isolation and properties of the specific cytoplasmic granules of

rabbit polyrnorphonuclear leucocytes. J. exp. Med. 112, 983-1004. COHNHEIM, J. (1867). Ueber Entziindung und Eiterung. Virchows Arch. 40, 1 7 9 . CORONADO, A., MARDONES, E. & ALLENDE, J. E. (1963). Isolation of hydroxylysyl-s-RNA and hydroxy-

prolyl-s-RNA in a chick embryo system. Biochem. biophys. Res. Commun. 13, 75-81. DANIELLI, J. F., FELL, H. B. & KODICEK, E. (1946). Some histological effects of partial deficiency of

vitamin C on healing processes : the influence on phosphatase formation in experimental skin wounds. Proc. Nutr. SOC. 4, 197-200.

Biochem. J . 43, 358-65.

saccharides in guinea-pig skin and cartilage. BY. J. Nutr. 11, 152-8.

48/80) on wound healing. J . Path. Bact. 78, 379-88.

C-deficient skin wounds. Biochem. J. 49, xvii-xviii.

taglio e delle perdite di sostanza. Annuli ital. Chir. 6,. 779-96.

307-42.

Proc. R. SOC. B 151, 517-36.

and hypodermis. Expl. Cell Res. 21, 394-405.

guinea pigs. Archs. Path. 49, 590-600.

rat. J. biophys. biochem. Cytol. g, 583-95.

DE DUVE, C. (1959). Subcellular particles, pp. 128-59. Ed. T. Hayashi. New York. DE DUVE, C. (1963). Ciba Foundation Symposium. Lysosomes, pp. 1-3 I. Ed. A. V. S. de Reuck and M. P.

DELAUNAY, A. & BAZIN, S. (1958). Le M6tabolisme du Collagkne. 111. Catabolisme et Turnover.

DE PETRIS, S., KARLSBAD, G. & PERNIS, B. (1962). Filamentous structures in the cytoplasm of normal

DI PASQUALE, G. & STEINETZ,B. G. (1964). Relationship of food intake to the effect of cortisone acetate on

DORFMAN, A. (1958). Studies on the biochemistry of connective tissue. Pediatrics 22, 576-89. DUNPHY, J. E. (1958). Repair of tissue after injury. Ann. N.Y. Acad. Sci. 73, 426-37.

Cameron. Boston.

Revue f r . gtzld. elin. biol. 3, 1101-j.

mononuclear phagocytes. J . Ultrastruct. Res. 7, 39-55.

skin wound healing. Proc. Sac. exp. Biol. Med. 117, 118-21.

The jibroblast and wound repair 83 DUNPHY, J. E. (1963). The fibroblast- :ubiquitous ally for the surgeon. New Engl. J . Med. 268,

DUNPHY, J. E. & UDUPA, K. N. (1955). Chemical and histochemical sequences in the normal healing of

DUNPHY, J. E., UDUPA, K. N. & EDWARDS, L. C. (1956). Wound healing. A new perspective with

EASTOE, J. E. (1955). The amino acid composition of mammalian collagen and gelatin. Biochem. J. 61, 589-600.

EDWARDS, L. C. & DUNPHY, J. E. (1958~) . Wound healing. I. Injury and normal repair. New Engl. J. Med. 259, 224-33.

EDWARDS, L. C. & DUNPHY, J. E. (19583). Wound healing. 11. Injury and abnormal repair. New Eng1.J. Med. 259, 275-85.

EDWARDS, L C., PERNOKAS, L. N. & DUNPHY, J. E. (1957). The use of a plastic sponge to sample regenerating tissue in healing wounds. Surgery Gynec. Obstet. 105, 303-9.

EISEN, A. Z. & GROSS, J. (1965). The role of epithelium and mesenchyme in the production of collagenase and hyaluronidase in the anuran tadpole. Devl Biol. 12, 408-18.

FAINSTAT, T. (1962). Hormonal basis for collagen bundle generation in uterine stroma: Extracellular studies of uterus. Endocrinology 71, 878-87.

FELL, H. B. & DANIELLI, J. F. (1943). The enzymes of healing wounds. I. The distribution of alkaline phosphomonoesterase in experimental wounds and burns in the rat. BY. J . exp. Path. 24, 196102.

FERNANDO, N. V. P. & MOVAT, H. Z. (1963). Fibrilogenesis in regenerating tendon. Lab. Invest. 12,

FEWER, D., THREADGOLD, J. & SHELDON, H. (1964). Studies on cartilage. V. Electron microscopic observations on the autoradiographic localization of Sg6 in cells and matrix. J. Ultrastruct. Res. 11,16672.

FISCHER, A. (1925). Sur la transformation in vitro des gros leucocytes mononuclkaires en fibroblastes. C . r. SOC. Biol., Paris 92, ~og-12.

FITTON JACKSON, S. (1955). Cytoplasmic granules in fibrogenic cells. Nature, Lond. 175, 39-40. FITTON JACKSON, S. (1956). The morphogenesis of avian tendon. Proc. R . SOC. B 14, 556-72. FITTON JACKSON, S. (1964). The cell. Vol. VI. Biochemistry, physiology, morphology, pp. 387-520. Eds.

J. Brachet and A. E. Mirsky. New York. FRENCH, J. E. & BENDITT, E. P. (1954). Observations on the localization of alkaline phosphatase in

healing wounds. Archs Path. 57, 352-6. FROMM, H. J. & NORDLIE, R. C. (1957). The healing of wounds, pp. 93-112, Ed. M. B. Williamson. New

York. GAINES, L. M., JR. (1960). Synthesis of acid mucopolysaccharides and collagen in tissue cultures of

fibroblasts. Johns Hopkins Hosp. Bull. 106, 195-204. GERBER, G., GERBER, G. & ALTMAN, K. I. (1960). Studies on the metabolism of tissue proteins. I.

Turnover of collagen labeled with proline-U-C14 in young rats. J. biol. Chem. 235, 2653-6. GERSH, I. & CATCHPOLE, H. R. (1949). The organization of ground substances and basement membrane

and its significance in tissue injury, disease and growth. Am. J . Anat. 85, 457-522. GIBEKING, R. (1959~) . Elektronenoptische Beobachtungen an Fibroblastenstruktur und Stoffwechsel

des Bindegewebes 11. Symposion an der Medizinischen Universitats-Klinik-Miinster pp. I 3 1-50. Eds. W. H. Hauss and H. Losse. Stuttgart.

GIESEKING, R. (19593). Uber die faserige Differenzierung des Mesenchyms in friihen Stadien der Ent- wicklung. Verh. dt. Ges. Path. 43, 56-60.

GIESEKING, R. ( I 963). Submikroskopische Strukturunterschiede zwischen Histiozyten und Fibro- blasten. Beitr. path. Anat. 128, 259-82.

GL~CKSMANN, A. (1964). Advances in biology of skin. Vol. v. Wound Healing, pp. 76-94. Eds. W Montagna and R. E. Billingham. London.

GLUCKSMANN, A., HOWARD, A. & PELC, S. R. (1956). The uptake of radioactive sulphate by cells, fibres and ground-substance of mature and developing connective tissue in the adult mouse. J. Anat. go, 478-85.

GODMAN, G. C. & LANE, N. (1964). On the site of sulfation in the chondrocyte. J . Cell Biol. 21, 353-66. GOLD, N. I. & GOULD, B. S. (195 I). Collagen fiber formation and alkaline phosphatase. Archs Biochem.

Biophys. 33, 155-64. GOLDBERG, B. & GREEN, H. (1964). An analysis of collagen secretion by established mouse fibroblast

lines. J. Cell Biol. 22, 227-58. GOULD, B. S. (1958). Biosynthesis of collagen. 111. The direct action of ascorbic acid on hydroxyproline

and collagen formation in subcutaneous polyvinyl sponge implants in guinea pigs. J. biol. Chem. 232, 637-50.

GOULD, B. S. (1963). Collagen formation and fibrogenesis with special reference to the role of ascorbic acid. Int. Rev. Cytol. 15, 301-61.

136777.

wounds. New Engl. J. Med. 253, 857-51.

particular reference to ascorbic acid deficiency. Ann. Surg. 14, 304-17.

214-29.

6-2

84 RUSSELL Ross GOULD, B. S., MANNER, G., GOLDMAN, H. M. & STOLMAN, J. M. (1960). Some aspects of collagen

formation. Ann. N . Y. Acad. Sci. 85, 385-98. GOULD, B. S. & WOESSNER J. F. (1957). Biosynthesis of collagen. The influence of ascorbic acid on the

proline, hydroxyproline, glycine, and collagen content of regenerating guinea pig skin. J . biol. Chem. 226, 289-300.

GRILLO, H. C. (1963). Origin of fibroblasts in wound healing: an autoradiographic study of inhibition of cellular proliferation by local X-irradiation. Ann. Surg. 157, 453-67.

GRILLO, H. C. & GROSS, J. (1959). Studies in wound healing. 111. Contraction in vit. C deficiency. Proc. SOC. exp. Biol. Med. 101, 268-70.

GRILLO, H. C. & GROSS, J . (1967). Collagenolytic activity during mammalian wound repair. Devl Biol. 15,300-17.

GRILLO, H. C. & POTSAID, M. S. (1961). Studies in wound healing: IV. Retardation of contraction by local X-irradiation, and observations relating to the origin of fibroblasts in repair. Ann. Surg. 154, 741-50.

GRILLO, H. C., WATTS, G. T. & GROSS, J. (1958). Studies in wound healing: I. Contraction and the wound contents, Ann. Surg. 148, 145-52.

GROSS, J. (1957). Studies on the fibrogenesis of collagen. Some properties of neutral extracts of connective tissue. Connective tissue, a symposium, pp. 45-61. Ed. R. E. Tunbridge. Oxford.

GROSS, J. (1958). Studies on the formation of collagen. I. Properties and fractionation of neutral salt extracts of normal guinea pig connective tissue. 3. exp. Med. 107, 247-64.

GROSS, J. (1959). Studies on the formation of collagen. IV. Effect of vitamin C deficiency on the neutral salt-extractable collagen of skin. J . exp. Med. 109, 55770.

GROSS, J . (1964). Organization and disorganization of collagen. Biophys. J . 4 (Suppl.), 63-78. GROSS, J., DUMSHA, B. & GLAZER, N. (1958). Comparative biochemistry of collagen. Biochim. biophys.

Acta 30, 2 9 3 7 . GROSS, J., HIGHBERGER, J. H. & SCHMITT, F. 0. (1955). Extraction of collagen from connective tissue by

neutral salt solutions. Proc. natn. Acad. Sci., U.S.A. 41, 1-7. GROSS, J. & KIRK, D. (1958). The heat precipitation of collagen from neutral salt solutions: some rate-

regulating factors. J. biol. Chem. 233, 355-60. GROSS, J. & LAPIERE, C. M. (1962). Collagenolytic activity in amphibian tissues: a tissue culture assay.

Proc. natn. Acad. Sci., U . S . A . 48, 1014-22. GUHA, A., BALASUBRAMANIAM, A. S., BASU, D . K., PATTABIRAMAN, T. N. & BACHHAWAT, B. K. (1961).

Metabolism of mucopolysaccharides during connective tissue formation. Symposium on proteins, pp. 187-93. Central Food Technological Research Institute, Mysore.

GUSTAVSON, K. H. (1956). The Chemistry and reactivity of collagen. New York. HARKNESS, R. D. (1955-6). Metabolism of collagen. Lect. scient. Basis Med. 5, 183-216. HARKNESS, R. D. (1961). Biological functions of collagen. Biol. Rev. 36, 399-463. HARKNESS, M. L. R., HARKNESS, R. D. & JAMES, F. W. (1958). The effect of a protein-free diet on the

collagen content of mice. J. Physiol., Lond. 4, 307-13. HARKNESS, R. D. & MORALEE, B. E. (1956). The time-course and route of loss of collagen from the rat’s

uterus during post-partum involution. J. Physiol., Lond. 132, 502-8. HARRINGTON, W. F. & VON HIPPEL, P. H. (1961). Advances in protein chemistry, Vol. XVI, pp. 1-138.

Eds. C. B. Anfinson, Jr., M. L. Anson, K. Bailey and J. Edsall. New York. HIGHBERGER, J. H., GROSS, J. & SCHMITT, F. (1951). The interaction of mucoprotein with soluble

collagen; an electron microscope study. Proc. natn. Acad. Sci., U . S . A . 37, 286-91. HIRSCH, J. G. (1965). The injlammatoryprocess, pp. 245-80. Eds. B. W. Zweifach, L. Grant and R. T.

McCluskey. New York. HODGE, A. J. , HICHBERGER, J. H. DEFFNER, G. J. 8~ SCHMITT, F. 0. (1960). The effects of proteases on

the tropocollagen macromolecule and on its aggregation properties. Proc. natn. Acad. Sci., U.S.A. 46, 197-206.

HOOKER, C. W. (1941). The healing of wounds of the uterus of the mouse. Anat. Rec. 79, 211-30. How=, E. L., BRIGGS, H., SHEA, R. & HARVEY, S. C. (1933). Effect of complete and partial starvation

HUNTER, J. (1812). A treatise on the blood, inflammation andgun shot wounds, p. 232. Pennsylvania. JACKSON, D . S. (1958). Some biochemical aspects of fibrogenesis and wound healing. New Engl. J . Med.

JACKSON, D. S . & BENTLEY, J. P. (1960). On the significance of the extractable collagens. J. biophys.

JACKSON, D. S. & FESSLER, J. H. (1955). Isolation and properties o f a collagen soluble in salt solution at

JACKSON, D. S., FLICKINGER, D. B. & DUNPHY, J. E. (1960). Biochemical studies of connective tissue

on the rate of fibroplasia in the healing wound. Archs Sztrg. 27, 846-58.

259, 814-20.

biochem. Cytol. 7, 37-42.

neutral pH. Nature, Lond. 176, 69-70.

repair. Ann. N . Y . Acad. Sci. 86, 943-7.

The jbroblast and wound repair 85 JACKSON, D. S., WATKINS, D. & WINKLER, A. (1964). Formation of s-RNA hydroxyproline in chick-

embryo and wound granulation tissue. Biochim. biophys. Acta 87, 152-3. JAMES, D. W. (1964). Biology of skin. Vol. V. Wound healing, pp. 216. Eds. W. Montagna & R. E. Billing-

ham. London. JAMIESON, J. D. & PALADE, G. E. (1966). Role of the Golgi complex in the intracellular transport of

secretory proteins. Proc. natn. Acad. Sci., U.S.A. 55, 424-31. JOHNSON, F. R. & MCMINN, R. M. H. (1958). The association of alkaline phosphatase with fibrino-

genesis. J . Anat. 92, 544-50. JOHNSON, F. R. & MCMINN, R. M. H. (1960). The cytology of wound healing of body surfaces in

mammals. Biol. Rev. 35, 364-412. JORGENSEN, 0. (1963~). The effect of thyroxine on wound healing in normal and ascorbic acid deficient

guinea pigs. Acta path. microbiol. scand. 59, 325-34. JORGENSEN, 0. (1963 b). A histological comparison of the effects of prednisone, phenylbutazone, and

sodium salicylate on granulation tissue formation in open wounds. Acta path. microbiol. scand. 59, 435-42.

JORGENSEN, 0. (1964~) . Electron-microscopical studies of granulation tissue formation of animals treated with antirheumatic compounds. Acta path. microbiol. scand. 60, 349-64.

JORGENSEN, 0. (1964b). Electron-microscopical studies of granulation tissue formation in open wounds of ascorbic acid deficient guinea pigs. Acta. path. microbiol. scand. 60, 36575.

JORGENSEN, L. & BORCHGREVINK, C. F. (I 964). The haemostatic mechanism in patients with haemorrhagic diseases. A histological study of wounds made for primary and secondary bleeding time tests. Acta path. microbiol. scand. 60, 55-82.

JUVA, K. & PROCKOP, D. (1964). Puromycin inhibition of collagen synthesis as evidence for a ribosomal or post-ribosomal site for the hydroxylation of proline. Biochim. biophys. Acta 91, 1 7 4 4 .

KAHLSON, G., NILSSON, K., ROSENGREN, E., ZEDERFELDT, B. & LUND, M. D. (1960). Wound healing. As dependent on rate of histamine formation. Lancet 11, 230-4.

KAJIKAWA, K., TANII, T. & HIRONO, R. (1959). Electron microscopic studies on skin fibroblasts of the mouse with special reference to the fibrillogenesis in connective tissue. Acta path. jap . 9, 61-80.

KAO, K. T. Y., HITT, W. E., BUSH, A. T. & MCGAVACK, T. H. (1964). Connective tissue. XII. Stimulat- ing effects of estrogens on collagen synthesis in rat uterine slices. Proc. SOC. exp. Biol. Med. 117,

KAO, K. Y. T., HITT, W. E. & MCGAVACK, T. H. (1965). Connective tissue. XIII. Effect of estradiol benzoate upon collagen synthesis by sponge biopsy connective tissue. Proc. SOC. exp. Biol. Med. 119, 3647.

KARFLER, H. E. (1960). Electron microscope study of developing chick embryo a0rta.J. Ultrastruct. Res. 4,

KELLY, E. W., JR. (1962~). Histology of sponge implant in the albino rat. Archs Path. 74, 542-50. KELLY, E. W., JR. (1962b). Estrogen effect on sponge implant histology in albino rat. Archs Path. 74,

KELSALL, M. A. (1961). Injlammation and diseases of connective tissue; a Hahnemann symposium, pp. 20-7.

KENNEDY, J. S. (1960). Sulphur-35 in connective-tissue formation. J. Path. Bact. 80, 359-72. KIAER, S. (1928). Biologische Beobachtung uber Wundheilung. Uber die Moglichkeit, den Wund-

heilungsprozeD mittels Huhnerembryonalsaft und Proteosen zu beschleunigen. Arch. klin. Chir. 149, I 46-8 2.

KIVIRIKKO, K. I. (1963). Hydroxyproline-containing fractions in normal and cortisone-treated chick embryos. Acta physiol. scand. 60 (Suppl.), 7-92.

KOBAK, M. W., BENDITT, E. P., WISSLER, R. W. & STEFFEE, C. H. (1947). The relation of protein deficiency to experimental wound healing. Surgery Gynec. Obstet. 85, 75 1-6.

KODICEK, E. & LOEWI, G. (1955). The uptake of sulphate by mucopolysaccharides of granulation tissue. Proc. R. Soc., Lond. IG, 100-15.

KOLLIKER, A. (1861). Neue Untersuchungen uber die Entwicklung des Bindgewebes. Wiirzb.-Natur- wissenschaften 2, 141-70.

KONNO, K. & TETSUKA, T . (1962). The isotopic studies on the conversion of proline to hydroxyproline. J. Biochem. 52, 466-7.

LEVENSON, S. M., BURKHILL, F. R. & WATERMAN, D. F. (1950). The healing of soft tissue wounds; the effects of nutrition, anemia, and age. Surgery 28, 905-35.

LEVINE, E. M., BECKER, Y., BOONE, c. W. & EAGLE, H. (1965). Contact inhibition, macromolecular synthesis, and polyribosomes in cultured human diploid fibroblasts. Proc. natn. Acad. Sci., U.S.A.

LIKAR, L., MASON, M. M. & ROSENKRANTZ, H. (1963). Response of the level of acid mucopolysaccharides

86-97.

420-54

551-3.

Eds. L. C. Mills and J. H. Moyer. Philadelphia, Pa.

53, 350-6.

in rat granulation tissue to cortisol. Endocrinology 72, 393-6.

86 RUSSELL Ross LIND, J. (1772). A treatise on scurvy. London. LINDSAY, W. K. & BIRCH, J. R. (1964). The fibroblast in flexor tendon healing. Plastic reconstr. Surg 34,.

LINDSTEDT, S . & PROCKOP, D. J. (1961). Isotopic studies on urinary hydroxyproline as evidence for rapidly catabolized forms of collagen in the young rat. J. biol. Chem. 236, 1399-403.

LOCALIO, S. A., MORGAN, M. E. & HINTON, J. W. (1948). The biological chemistry of wound healing. I. The effect of dl-methionine on the healing of wounds in protein-depleted animals. Surgery G'ynec. Obstet. 86, 582-90.

LOWTHER, D. A., GREEN, N. M. & CHAPMAN, J. A. (1961). Morphological and chemical studies of collagen formation. 11. Metabolic activity of collagen associated with subcellular fractions of guinea- pig granulomata. J. biophys. biochem. Cytol. 10, 373-88.

LUCCIOLI, G. M., KAHN, D. S. & ROBERTSON, H. R. (1964). Histologic study of wound contraction in the rabbit. Ann. Surg. 160, 1030-40.

LUKENS, L. N. (1965). Evidence for the nature of the precursor that is hydroxylated during the biosynthesis of collagen hydroxyproline. J. biol. Chem. 240, 1661-9.

MACDONALD, R. A. (I 959). Origin of fibroblasts in experimental healing wounds : autoradiographic studies using tritiated thymidine. Surgery 46, 376-82.

MCMINN, R. M. H. (1960). Cellular anatomy of experimental wound healing. Ann. R. Coll. Surg. 26, 245.

MANCINI, R. E., VILAR, O., STEIN, E. & FIORINI, H. (1961). A histochemical and radioautographic study of the participation of fibroblasts in the production of mucopolysaccharides in connective tissue. J. Histochem. Cytochern. 9, 278-91.

MANNER, G. & GOULD, B. S. (1962). Collagen biosynthesis. The formation of an s-RNA-hydroxyproline complex. Fedn Proc. Fedn Am. Sots. exp. Biol. 21, 169.

MANNER, G. & GOULD, B. S. (1963). Collagen biosynthesis. The formation of hydroxyproline and soluble-ribonucleic acid complexes of proline and hydroxyproline by chick embryo in vivo and by a subcellular chick-embryo system. Biochim. biophys. Actu 72, 243-50.

MARTIN, C. J. & AXELROD, A. E. (1953). A modified method for determination of hydroxyproline. Proc. SOC. exp. Biol. Med. 83,461-2.

MAXIMOW, A. (I 927). Development of non-granular leucocytes (lymphocytes and monocytes) into polyblasts (macrophages) and fibroblasts in vitro. Proc. SOC. exp. Biol. Med. 24, 570-2.

MAXIMOW, A. (1928). Cultures of blood leucocytes. From lymphocyte and monocyte to connective tissue. Arch. exp. Zellforsch. 5, 169-268.

MERKER, H. J. (196 I). Elektronenmikroskopische Untersuchungen uber die Fibrillogenese im mensch- lichen Granulationsgewebe. Langenbecks Arch. klin. Chir. 297, 41 1-23.

MEYER, K., HOFFMAN, P. & LINKER, A. (1957). The acid mucopolysaccharides of connective tissue. In Connectiwe tissue. A symposium, pp. 86-96. Ed. R. E. Tunbridge. Oxford.

MILLER, L. & WHITTING, H. W. (1965). Mast cells and wound healing of the skin in the rat. Z . Zellforsch. mikrosk. Anat. 65, 597406.

MITOMA, C. & SMITH, T. E. (1960). Studies on the role of ascorbic acid in collagen synthesis. J. biol. Chem. 235,426-8.

MOEN, J. K. (1935). The development of pure cultures of fibroblasts from single mononuclear cells. J. exp. Med. 61, 247-60.

MONIS, B. (1963). Variations of aminopeptidase activity in granulation tissue and in serum of rats during wound healing. A correlated histochemical and quantitative study. Am. J. Path. 42,

MONIS, B., NACHLAS, M. M. & SELIGMAN, A. M. (1959). Study of leucine aminopeptidase in neoplastic

MONTAGNA, W. & HILL, C. R. (1957). The localization of Sa6 in the skin of the rat. Anat. Rec. 127,

MORGAN, C . F. (1963). A study of estrogenic action on the collagen, hexosamine and nitrogen content

MOTTET, N. K. (1961). Histochemical observations on the role of stromal aminopeptidase activity follow-

MOVAT, H. Z . & FERNANDO, N. V. P. (1962). The fine structure of connective tissue. I. The fibroblast.

MUIR, H. (I 961). Chondroitin sulphates and sulphated polysaccharides of connective tissue. Biochem.

NEEDHAM, A. E. (1952). Regeneration and wound-healing. London. NEEDHAM, A. E. (1964). Advances in biology of skin. Vol. v. Wound healing, pp. 1-29. Eds. W. Montagna

NEUBERGER, A. (1955). Metabolism of collagen under normal conditions. S.E.B. Symposia g, 72-84.

223-32.

301-14.

and inflammatory tissues with a new histochemical method. Cancer 12, 601-8.

163-72.

of skin, uterus and vagina. Endocrinology 73, I 1-19.

ing neoplastic cell (H. Ep. no. 3) invasion. Am. J. Path. 39, 17-26

Expl. Molec. Path. I, 509-39.

SOC. Symp. 20, 4-22.

and R. E. Billingham. London.

The Jibroblast and wound repair 87 NEUBERGER, A. & SLACK, H. G. B. (1953). The metabolism of collagen from liver, bone, skin and tendon

NEUMAN, R. E. & LOGAN, M. A. (1949). The determination of hydroxyproline. J. biol. Chem. 184,

NEUMAN, R. E. & LOGAN, M. A. (1950). The determination of collagen and elastin in tissues. J. biol. Chem. 186, 549-56.

NEUTRA, M. & LEBLOND, C. P. (1966). Radioautographic comparison of the uptake of galactose-H3 in the Golgi region of various cells secreting glycoproteins or mucopolysaccharides. J. Cell Biology 30,

NOCFXTI, M. R., LEDERMAN, G. E., FUMY, C. A. & LOPANO, A. J. (1964). Collagen synthesis and C1*- labeled proline uptake and conversion to hydroxyproline in steroid-treated granulomas. Proc. SOC. exp. Biol. Med. 117, 215-18.

OPIE, E. L. (1922). Intracellular digestion. The enzymes and anti-enzymes concerned. Physiol. Rew. 2,

552-85. PALADE, G. E. (1955). Relations between the endoplasmic reticulum and the plasma membrane in macro-

phages. Anat. Rec. 131, 445. PALADE, G. E. (1956). The endoplasmic reticulum. J . biophys. biochem. Cytol. (Suppl.), 2, 85-98. PALADE, G. E. (1958a). Frontiers in cytology, pp. 283-304. Ed. S. L. Palay. New Haven, Conn. PALADE, G. E. (19586). Microsomal particles and protein synthesis, pp, 36-61. Ed. R. B. Roberts. New

PALADE, G. E. & PORTER, K. R. (1954). Studies on the endoplasmic reticulum. I. Its identification in

PALADE, G. E. & SIEKEVITZ, P. (rg56a). Liver microsomes. An integrated morphological and biochemical

PALADE, G. E. & SIEKEVITZ, P. (19566). Pancreatic microsomes. An integrated morphological and bio-

PEACH, R. (1962). An electron optical study of experimental scurvy. 3. Ultrastruct. Res. 6, 579-90. PEACH, R., WILLIAMS, G. & CHAPMAN, J. A. (1961). A light and electron optical study of regenerating

tendon. Am. J. Path. 38, 495-513. PENNEY, J. R. & BALFOUR, B. M. (1949). The effect of vitamin C on mucopolysaccharide production in

wound healing. J. Path. Bact. 61, 171-8. PEREZ-TAMAYO, R. & IHNEN, M. (1953). The effect of methionine in experimental wound healing. A

morphologic study. Am. J. Path. zg, 233-50. PERNOKAS, L. N., EDWARDS, L. C. & DUNPHY, J. E. (1957). Hormonal influence on healing wounds: the

effect of adrenalectomy and cortisone on the quantity and collagen content of granulation tissue. Surg. Forum 8, 75-6.

PERSON, B. H. (1953). Studies on connective tissue ground substance. I. Histochemical features of ground substance mucopolysaccharides. 11. Organization of the ground substance in ascorbic acid deficiency and its modification by the action of cortisone. Acta SOC. Med. upsal. 58 (Suppl.) 1-119.

PETERKOFSKY, B. & UDENFRIEND, S. (1961). Conversion of proline-C1' to peptide-bound hydroxyproline-C'* in a cell-free system from chick embryo. Biochem. biophys. Res. Commun. 6, 184-90.

PETERKOFSKY, B. & UDFNFRIEND, S. (1963). Conversion of proline to collagen hydroxyproline in a cell- free system from chick embryo. J. biol. Chem. 238, 396677.

PETERKOFSKY, B. & UDENFRIEND, S. (1965). Enzymatic hydroxylation of proline in microsomal polypeptide leading to formation of collagen. Proc. natn. Acad. Sci., U.S.A. 53, 335-42.

PETERSON, M. & LEBLOND, C. P. (1964). Synthesis of complex carbohydrates in the Golgi region, as shown by radioautography after injection of labelled glucose. J. Cell Biology 21, 143-8.

PETRAKIS, N. L. (1961). In wiwo cultivation of leukocytes in diffusion chambers: requirement of ascorbic acid for differentiation of mononuclear leukocytes to fibroblasts. Blood. 18, 310-1 6.

PETRAKIS, N. L., DAVIS, M. & LUCIA, S. P. (1961). The in wivo differentiation of human leukocytes into histiocytes, fibroblasts and fat cells in subcutaneous diffusion chambers. Blood 17, 109-18.

PIEZ, K. A. (1960). The relation between amino acid composition and denaturation of vertebrate collagens. J. Am. chem. Sac. 82, 247.

PIEZ, K. A. (1964). Nonidentity of the three a chains in codfish skin collagen. J. biol. Chem. 239,

PIEZ, K. A., EIGNER, E. A. & LEWIS, M. S. (1963). The chromatographic separation and amino acid

PIEZ, K. A. & GROSS, J. (1959). The amino acid composition and morphology of some invertebrate and

PIEZ, K. A., LEWIS, M. S. MARTIN, G. R. & GROSS, J. (1961). Subunits of the collagen molecule.

in the normal rat. Biochem. J. 53, 47-52.

299-306.

137-50-

York.

cells in situ. J . exp. Med. 100, 641-56.

study. J. biophys. biochem. Cytol. 2, 171-200.

chemical study. J. biophys. biochem. Cytol. 2, 671-90.

P c 4 3 15-6.

composition of the subunits of several collagens. Biochemistry 2, 58-66.

vertebrate collagens. Biochim. biophys. Acta 34, 24-39.

Biochim. biophys. Acta 53, 596-8.

88 RUSSELL Ross PI=, K. A. & LIKINS, R. C. (1957). The conversion of lysine to hydroxylysine and its relation to the

biosynthesis of collagen in several tissues of the rat. J . biol. Chem. 229, 101-10. PIRANI, C. L. & LEVENSON, S. M. (1953). Effect of vitamin C deficiency on healed wounds. Proc. SOC.

exp. Biol. Med. 83, 95-9. PORTER, K. R. (1951). Repair processes in connective tissue, interrelations with special reference to

calcium. In Connective tissue. Ed. E. C. Reifenstein, Jr.. p. 126. Trans. 4th Con.. ofJosiah MacyJr . Found.

PORTER, K. R. (1953). Observations on a submicroscopic basophilic component of cytoplasm. J. exp. Med. 97, 727-50.

PORTER, K. R. (1961). The cell; biochemistry, physiology, morphology. Vol. 11, pp. 621475. Eds. J. Brachet and A. E. Mirsky. New York.

PORTER, K. R. (1964). Cell fine structure and biosynthesis of intercellular macromolecules. Biophys. J.

PORTER, K. R. & PAPPAS, G. D. (1959). Collagen formation by fibroblasts of the chick embryo dermis. J. biophys. biochem. Cytol. 5, 15346.

POSTLETHWAIT, R. W., CHEN, R. W., KAMIN, H. & DILLON, M. L. (1960). The distribution of radioactive ascorbic acid in wounded normal and scorbutic guinea pigs. Surgery 47, 417-19.

PROCKOP, D. J. & JUVA, K. (1965). Synthesis of hydroxyproline in vitro by the hydroxylation of proline in a precursor of collagen. Proc. natn. Acad. Sci., U.S.A. 53, 661-8.

PROCKOP, D. J., PETERKOFSKY, B. & UDENFRIEND, S. (1962). Studies on the intracellular localization of collagen synthesis in the intact chick embryo. J. biol. Chetp. 237, 1581-4.

RAEKALLIO, J. (1960). Enzymes histochemically demonstrable in the earliest phase of wound healing. Nature, Lond. 188, 234-5.

RAEKALLIO, J. (I 961). Histochemical studies on vital and post-mortem skin wounds. Experimental investigation on medicolegally significant vital reactions in an early phase of wound healing. Annls Med. exp. Biol. Fenn. 39 (Suppl. 6), 105 pages.

RAEKALLIO, J. (1963 a). Histochemical demonstration of monoamine oxidase in the earliest phase of wound healing. Nature, Lond. 199, 4967.

RAEKALLIO, J. (1963 b). P-Glucuronidase activity in the initial phase of wound healing. Naturwissm- schaften 50, 596.

RAEKALLIO, J. & LEVONEN, E. (I 963 a). Histochemical demonstration of trans-glucosylases and glycogen in the 'lag' phase of wound healing. Expl Molec. Path. 2, 69-73.

RAEKALLIO, J. & LEVONEN, E. (1963 b). The appearance of esterases in rat skin wounds. Annls Med. exp. Biol. Fenn. 41, 305-10.

RAEKALLIO, J. & LEVONEN, E. (1963~). Adenosine triphosphatase activity of rat skin in early wound healing. Acta path. microbiol. scand. 58, 451-6.

RAEKALLIO, J., LINDFORS, R., ELFVING, G., HXSTBACKA, J. & PUITTINEN, J. (1964). Histochemical observations onwound healing in denervated and healthy rat skin. Actapath. microbiol. scand. 62,53-8.

RAGAN, C., HOWES, E. L., PLOTZ, C. M., MEYER, K. & BLUNT, J. W. (1949). Effect of cortisone on production of granulation tissue in the rabbit. Proc. SOC. exp. Biol. Med. 72,718-21.

RAMACHANDRAN, G. N. (1963). Molecular structure of collagen. In International Rewiew of Connective Tissue Research, Vol. I, pp. 127-82. Ed. D. Hall. New York.

RAMANATHAN, N. (1962). C08agen. New York. REMAK, R. (I 852). Extracellulare Entstehung thierischer Zellen und uber Vermehrung derselben durch

Theilung. Arch. Anat. Phys. Wissensch. Med. I, 47-57. REVEL, J.-P. & HAY, E. L. (1963). An autoradiographic and electron microscopic study of collagen

synthesis in differentiating cartilage. Z. Zellforsch. mikrosk. Anat. 61, I 1-44. RIDDLE, J. M. & BARNHART, M. I. (1964). Ultrastructural study of fibrin dissolution via emigrated

polymorphonuclear neutrophils. Am. J . Path. 45, 805-24. RIDDLE, J. M. & BARNHART, M. I. (1965). The eosinophil as a source for profibrinolysin in acute

inflammation. Blood 25, 776-94. ROBERTSON, W. VAN B. (1952). Influence of ascorbic acid on NlS incorporation into collagen in vivo.

J. biol. Chem. 197, 495-504. ROBERTSON, W. VAN B. & HEWITT, J. (1961). Augmentation of collagen synthesis by ascorbic acid

in vitro. Biochim, biophys. Acta 49, 404-6. ROBERTSON, W. VAN B. & HINDS, H. (1956). Polysaccharide formation in repair tissue during ascorbic

acid deficiency. J . biol. Chem. 221, 791-6. ROBERTSON, W. VAN B., HIWEIT, J. & HERMANN, C. (1959). The relation of ascorbic acid to the con-

version of proline to hydroxyproline in the synthesis of collagen in the carrageenan granuloma. J. biol. Chem. 234, 105-08.

ROBERTSON, W. VAN B. & SCHWARTZ, B. (1953). Ascorbic acid and the formation of collagen. J. biol. Chem. 201, 689-96.

4,167-96.

The Jibroblast and wound repair ss Ross, R. (1964). Biology of skin. Vol. v. Wound healing, pp. 144-64. Eds. W. Montagna and R. E

Billingham. London. Ross, R. & BENDITT, E. P. (1961). Wound healing and collagen formation. I. Sequential changes in

components of guinea pig skin wounds observed in the electron microscope. J . biophys. biochem. Cytol. XI, 677700.

Ross, R. & BENDITT, E. P. (1962~). Wound healing and collagen formation. 11. Fine structure in experimental scurvy. J. Cell Biol. 12, 533-51.

Ross, R. & BENDITT, E. P. (1962b). Wound healing and collagen formation. 111. A quantitative radioautographic study of the utilization of proline-H8 in wounds from normal and scorbutic guinea pigs. J. Cell Biol. 15, 99-108.

Ross, R. & BENDITT, E. P. (1964). Wound healing and collagen formation. IV. Distortion of ribosomal patterns of fibroblasts in scurvy. J. Cell Biol. 22, 365-90.

Ross, R. & BENDITT, E. P. (1965). Wound healing and collagen formation. V. Quantitative electron microscope radioautographic observations of proline-H8 utilization by fibroblasts. J. Cell Biol. 27, 83-106.

Ross, R. & LILLYWHITE, J. W. (1965). The fate of buf€y coat cells grown in subcutaneously implanted diffusion chambers. A light and electron microscopic study. Lab. Invest. 14, 1568-85.

SANDBERG, N. (1964~). Enhanced rate of healing in rats with an increased rate of histamine formation. Acta chir. scand. 127, 9-21.

SANDBERG, N. (1964b). Granulation tissue hydroxyproline in the rat after inhibition of histamine formation. Acta chir. scand. 127, 22-34.

SANDBERG, N. (1964~). Time relationship between administration of cortisone and wound healing in rats Acta chir. scand. 127, 446-55.

SANDBERG, N. & STEINHARDT, C. (1964). On the effect of cortisone on the histamine formation of skin wounds in the rat. Acta chir. scand. 127, 5747.

SAVLOV, E. D. & DUNPHY, J. E. (1954). The healing of the disrupted and resutured wound. Surgery 26, 362-70.

SCHAUBLE, J. F., CHEN, R. POSTLETHWAIT, R. W. & DILLON, M. L. (1960). A study of the distribution of ascorbic acid in the wound healing of guinea pig tissue. Surgery Gynec. Obstet. 110, 314-8.

SCHILLER, S., BLUMENKRANTZ, N., & DORFMAN, A. (1965). Effect of prednisolone and hydrocortisone on distribution of mucopolysaccharides in the skin of rats. Biochim. biophys. Acta 101,

135-7. SCHILLER, S. & DORFMAN, A. (1957). The metabolism of mucopolysaccharides in animals: The effect

of cortisone and hydrocortisone on rat skin. Endocrinology 60, 376-81. SCHILLING, J. A., FAVATA, B. V. & RADAKOVITCH, M. (1953). Studies of fibroplasia in wound. Surgery

Gynec. Obstet. 96, 143-9. SCHULTZE, M. S. (1861). Uber Muskelkorperchen und das, was man eine Zelle zu nennen habe. Arch.

Anat. Physiol. Wiss. Med. I , 1-27. SCHWA", T. (1847). Microscopical researches into the accordance in the structure andgrowth of animals

and plants. p. 45. Trans. by H. Smith. Sydenham Society, London. SELYE, H. (1953). Part of inflammation in local adaptation syndrome. In Symposium on the mechanism of

inJammation, p. 53. Eds. G. Jasmin and A. Robert. Montreal. SHELDON, H. & KIMBALL, F. B. (1962). Studies on cartilage. 111. The occurrence of collagen within

vacuoles of the Golgi apparatus. J. Cell. Biology 12, 599-613. SHETLAR, M. R., LACEFIELD, E. G., WHITE, B. N. & SCHILLING, J. A. (1959). Wound healing: glyco-

proteins of wound tissue. I. Studies of hexosamine, hexose, and uronic acids content. Proc. SOC. exp. Biol. Med. 100, 501-3.

SINEX, F. M. & VAN SLYXE, D. D. (1955). The source and state of the hydroxylysine of collagen. J. biol. Chem. 216, 245-50.

SINEX, F. M., VAN SLYKE, D. D. & CHRISTMAN, D. R. (1959). The source and state of the hydroxylysine of collagen. 11. Failure of free hydroxylysine to serve as a source of the hydroxylysine or lysine of collagen. J. biol. Chem. 234, 918-21.

SLACK, H. G. B. (1958). Connective tissue growth stimulated by carrageenin. 3. The nature and amount of polysaccharide produced in normal and scorbutic guinea pigs and the metabolism of a chondroitin sulphuric acid fraction. Biochem. J. 69, 125-34.

SMITH, 0. W. & KALTREIDER, N. B. (1963). Collagen content of the nonpregnant rat uterus as related to the functional responses to estrogen and progesterone. Endocrinology 73, 6 19-28.

SPAIN, D. M., MOLOMUT, N. & HABER, A. (1952). Studies of the cortisone effects on the inflammatory response. I. Alterations of the histopathology of chemically induced inflammation. J. Lab. clin. Med.

STAHL, S . S. (1962). The effect of a protein-free diet on the healing of gingival wounds in rats. Archs 39, 383-9.

Oral Biol. 7, 551-6.

90 RUSSELL Ross STAHL, S. S. (1963). Healing of gingival wounds in female rats fed a low-protein diet. J. dent. Res. q2,

STEARNS, M. L. (1940U). Studies on the development of connective tissue in transparent chambers in

STEARNS, M. L. (19406). Studies on the development of connective tissue in transparent chambers in

STEHBENS, W. E. (1962). The production of sulphated mucopolysaccharides by endothelium. J. Path.

1511-6.

the rabbit’s ear. I. Am. J. Anat. 66, 133-76.

the rabbit’s ear. 11. Am. J. Anat. 67, 55-97.

Bact. 83, 337-46. STERN, K. G. (1932). uber die Katalase farbloser Blutzellen. Hoppe-Seyler’s 2. physiol. Chem. 204,

259-82. STETTEN, M. R. (1949). Some aspects of the metabolism of hydroxyproline, studied with the aid of

isotopic nitrogen. J. biol. Chem. 181, 31-8. STETTEN, M. R. & SCHOENHEIMER, R. (1944). The metabolism of I(-)-proline studied with the aid of

deuterium and isotopic nitrogen. J. biol. Chem. 153, I 13-32. STONE, N. & MEISTER, A. (1962). Function of ascorbic acid in the conversion of proline to collagen

hydroxyproline. Nature, Lond. 194, 5 5 5 7 . TAUBENHAUS, M. (1953). The influence of cortisone upon granulation tissue and its synergism and

antagonism to other hormones. Ann. N. Y. Acad. Sci. 56, 666-73. TAUBENHAUS, M., JACOBSON, M., MORTON, J. V. & LEVINE, R. (1953). Parallel inhibition of granul-

ation tissue by cortisone, hydrocortisone, dibenamine and banthine. Proc. SOC. exp. Biol. Med. 84,

THOMPSON, R. C. & BALLOU, J. E. (1956). Studies of metabolic turnover with tritium as a tracer. V. The predominantly nondynamic state of body constituents in the rat. J. biol. Chem. 223,

UDUPA, K. N., WOESSNER, J. F. & DUNPHY, J. E. (1956). The effect of methionine on the production of mucopolysaccharides and collagen in healing wounds of protein-depleted animals. Surgery Gynec.

646-5 I .

795-810.

Obstet. 102, 639-45.

Uptake in healing wounds, megakaryocytes, and blood platelets. Archs. Path. 62, 194-9. UPTON, A. C. & ODELL, T. T., JR. (1956). Utilization of Sa6-labeled sulfate in scorbutic guinea pigs.

U R I ~ Z K Y , M., F ~ I , J. M.; & M E I L G , E; (1965). Cefi-free collagen biosynthesis andthe hydroxy-

VAES, G. M. & NICHOLS, G., JR. (1962). Metabolism of glycine-1-P by bone in vitro: effects of hor-

VILJANTO, J. (1964). Biochemical basis of tensile strength in wound healing. An experimental study with

VIRCHOW, R. (1852). Einige neue Beobachtungen mit uber Knochen und Knorpelkorperchen. Verh. dt.

lation of sRNA-proline. Archs. Biochem. Biophys. 109, 480-9.

mones and other factors. Endocrinology 70, 890-901.

viscose cellular sponges on rats. Acta chir. scand. (Suppl. 333), IOI pages. . - .

Ges. Path. I, 13. VOGEL, A. (I 964). Feinstrukturelle Charakteristika von Kollagenfasern. Pathologia Microbiol. 27,

436-46. VOLKMAN, A. & GOWANS, J. L. (1965 a). The production of macrophages in the rat. Br. J . exp. Path. 46,

VOLKMAN, A. & GOWANS, J. L. (19566). The origin of macrophages from bone marrow in the rat. Br.

WASSERMANN, F. (1954). Fibrillogenesis in the regenerating rat tendon with special reference to growth

WASSERMANN, F. ( I 956). V. The intercellular components of connective tissue : origin, structure and

WASSERMANN, F. & KUBOTA, L. (1956). Observations on fibrillogenesis in the connective tissue of the

WEISS, P. (1959). The biological foundations of wound repair. Harvey Lect. 55, 13-42. WEISS, P. & GAMIER, B. (1952). Shape and movement of mesenchyme cells as functions of the physical

structure of the medium. Contributions to a quantitative morphology. Proc. natn. Acad. Sci., U.S.A.

50-61.

J . exp. Path. 46, 62-70.

and composition of the collagenous fibril. Am. J . Anat. 94, 399-438.

interrelationship of fibers and ground substance. Ergebn. Anat. EntwGesch. 35, 240-330.

chick embryo with the aid of silver impregnation. J . biophys. biochem. Cytol. 2 (Suppl.), 67-72.

. _ _ 38, 246-80.

the healing of wounds. Ann. N. Y. Acad. Sci. 94, 297-307. WHITE, B. N., SHETLAR, M. R. & SCHILLING, J. A. (1961). The glycoproteins and their relationship to

WHITE, B. N., SHETLAR, M. R., SHURLEY, H. Mi & SCHILLING, J. A. (1965). Incorporation of (I-14C) glucosamine into mucopolysaccharides of rat connective tissue. Biochim. biophys. Acta 101, 97-105.

WICHMANN, B. E. (1955). The mast cell count during the process of wound healing. An experimental investigation on rats. Acta path. microbiol. scand. (Suppl. I O ~ ) , 35 pages.

WILLIAMS, G. (1957). A histological study of the connective-tissue reaction to carrageenin. J . Path.

WILLIAMSON, M. B. (1956). Protein metabolism during healing of wounds. Clin. Chem. 2, 1-15. Bact. 73, 557-64.

The Jibroblast and wound repair 91 WILLIAMSON, M. B. (1957). Metabolism of proteins and amino acids during wound healing. In The

healing of wounds. A symposium on recent trends and studies, pp. 1-27. Ed. M. B. Williamson. New York.

WILLIAMSON, M. B. & FROMM, H. J. (1953). Utilization of sulphur amino acids during healing of experimental wounds. Proc. SOC. exp. Biol. Med. 83, 329-333.

WILLIAMSON, M. B. & FROMM, H. J. (1955). The incorporation of sulphur amino acids into the proteins of regenerating wound tissue. J . biol. Chem. 212. 705-12.

WILLIAMSON, M. B., GAGAN, J. F., HALEY, H. B. &WILLIAMSON, D. A. (1965). Rateof incorporation of methionine-Sa6 into the regenerating tissue of experimental wounds. J. surg. Res. 5, 146-9.

WILLIAMSON, M. B. & GUSCHLBAUER, W. (1961). Metabolism of nucleic acids during regeneration of wound tissue. J. biol. Chem. 236, 1463-6.

WILLIAMSON, M. B., MCCARTHY, T. H. & FROMM, H. J. (1951). Relation of protein nutrition to the healing of experimental wounds. Proc. SOC. exp. Biol. Med. 77, 302-5.

WILLMER, E. N. & JACOBY, F. (1936). Studies on growth of tissues in vitro: on the manner in which growth is stimulated by extracts of embryo tissues. J. exp. Biol. 13, 237-48.

WILLSTATTER, R., BAMANN, E. & ROHDEWALD, M. (1930). Uber das Trypsin der farblosen Blut- korperchen. Fiinfte Abhandlung iiber Enzyme der Leukocyten. Hoppe-Seyler’s 2. Physiol. Chem. 188, 107-23.

WINDRUM, G. M. (1958). Sulfation technics in the histologic study of granulation tissue. Lab. Invest. 7, 9-18.

WOESSNER, J. F., JR & BOUCEK, R. J. ( 1 9 6 1 ~ ) . Connective tissue development in subcutaneously implanted polyvinyl sponge. I. Biochemical changes during development. Archr Biochem. Biophys. 93, 85-94.

WOESSNER, J. F., JR. & BOUCEK, R. J. (1961b). Connective tissue development in subcutaneously implanted polyvinyl sponge. 11. Enzymic changes during development. Archs Biochem. Biophys. 93, 95-109.

WOLBACH, S. B. (1933). Controlled formation of collagen and reticulum. A study of the source of intercellular substance in recovery from experimental scorbutus. Am. J . Path. 9, 689-700.

WOLBACH, S. B. & BESSEY, 0. A. (1942). Tissue changes in vitamin deficiencies. Physiol. Rev. 22,

WOLBACH, S. B. & HOWE, P. R. (1926). Intercellular substances in experimental scorbutus. Archs. Path. Lab. Med. I, 1-24.

YARDLEY, J. H., HEATON, M. W., GAINES, L. M., JR. & SHULMAN, L. E. (1960). Collagen formation by fibroblasts. Preliminary electron microscopic observations using thin sections of tissue cultures. Johns Hopkins Hosp. Bull. 106, 381-93.

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