Journal of Medical and Biological Engineering, 29(6): 304-310 304

Guided Tissue Regeneration with Use of CaSO4-Chitosan

Composite Membrane

Shyh-Ming Kuo Gregory Cheng-Chie Niu Cheng-Wen Lan Min-Feng Cheng

Min-Yu Chiang Shwu-Jen Chang*

Department of Biomedical Engineering, I-Shou University, Kaohsiung 840, Taiwan, ROC

Received 7 Jul 2009; Accepted 10 Oct 2009

Abstract

Guided tissue regeneration (GTR) membranes with bioresorbable characteristics have been employed in recent

years for periodontal procedures to deflect the growth of gingival tissues away from root surface. They provide an

isolated space over regions with defective tissues and allow the relatively slow-growing periodontal ligament

fibroblasts to be repopulated over the root surface. In this study, we employed chitosan and CaSO4 composites as one

of the viable membrane materials and evaluated their roles in GTR applications. Two types of CaSO4-chitosan

composite membranes with molecular weights of chitosan 70 kDa and 300 kDa, respectively, were prepared for study

in three categories: mechanical strength to create and sustain an effective space; rapid rate to reach hydrolytic

equilibrium in phosphate buffer solution; and ease of clinical manipulations. Consequently, standardized, trans-osseous

and critical-sized (cavity of 6 mm) skull defects were made in adult rats, and the defective regions were covered with

the specifically prepared chitosan membranes. After 4 weeks of recovering, varying degrees of bone healing were

observed beneath the CaSO4-chitosan composite membranes in comparison to the control group. The CaSO4-chitosan

composite membrane-covered regions showed a clear boundary space between connective tissues and bony tissues. In

summary, good cell-occlusion and beneficial osteogensis effects by these bioresorbable GTR materials toward the

wound recovery were indicated.

Keywords: Guided tissue regeneration (GTR), Chitosan, CaSO4, Membrane

1. Introduction

Guided tissue regeneration (GTR) techniques have been

successfully applied in the treatment of periodontal diseases

and provided opportunity for formation of new bone [1-3]. The

techniques involved procedures employing membranes as

mechanical barriers to create a space around the defects that

might permit bone regeneration and to prevent the epithelial

cells from migrating into the bony area. In general, an effective

barrier membrane should have appropriate mechanical strength

to occlude the rapid growth of repairing tissues (epithelium and

gingival connective tissue) away from the root surface and

maintain a protective space over the defect that will allow

migration of bone cells from the surrounding alveolar bone into

the targeted area. Ideally, clinical manageability of the barrier

membrane is also a necessary consideration during surgical

procedures. In addition, effective materials should be safe,

non-toxic, and non-antigenic ones which induce little or no

* Corresponding author: Shwu Jen Chang

Tel: +886-7-6577711 ext. 6719; Fax: +886-7-6577067

E-mail: sjchang@isu.edu.tw

inflammatory responses around the host tissue. Furthermore,

the barrier function must be established and maintained for a

period of time long enough for tissue guidance to take effect.

As a result, extensive effort has been employed to utilize

bio-resorbable membranes to achieve therapeutic purpose in

clinical trials [4-7].

Among the bio-resorbable materials used in GTR

applications, resorbable membranes such as collagen

membranes and polyglycolic acid-based membranes, have been

successfully applied and are commercially available. However,

there could be disadvantages in the use of collagen membrane

because it might cause localized chronic inflammatory response

and rapid degradation behavior [8,9]. As for the use of

polyglycolic acid-based membrane, there could be problems

such as gingival recession, exposure of the device, and

inflammation of surrounding soft tissue [10,11].

Chitosan, a natural occurring polysaccharide, is widely

present among marine and terrestrial invertebrates and lower

forms of the plant kingdom. Highly deacetylated chitosan (e.g.,

> 85%) exhibits low degradation rate in aqueous media and

may last several months, and thus leads to great potential in the

development of inexpensive and versatile drug encapsulating

J. Med. Biol. Eng., Vol. 29. No. 6 2009 305

systems [12,13]. Its excellent gel-forming properties and ability

to be re-shaped into various forms simply by thermally induced

separation method strongly enhances its potential applications

in the biomedical field. Many researchers also found that

chitosan has osteoinduction and osteoconduction potentials

when used as a bone scaffold material [14,15]. As a

biodegradable material, chitosan had been given a lot of

attention in biomedical applications. Its blood clotting

characteristics made it well accepted as a wound healing agent,

skin grafting template and hemostatic agent [16-18]. In general,

it is also well-known to promote the formation of scaffolding

matrix in tissue regeneration process. On the other hand, among

many commercially available ceramics, CaSO4 is frequently

used as bone repairing and replacement material because of its

biocompatibility, bioactivity, mechanical strength, and

non-toxicity. From clinical experience, CaSO4 can be gradually

absorbed; that is followed by new bone formation yet without

compromising the required intimacy of bone-implant contact.

Kinetically, CaSO4 degraded faster than tricalcium phosphates

and have shown favorable osteoconduction and resorption

properties in animal studies. Also, CaSO4 can be resorbed and

replaced by new remodeled bone completely [19,20].

Consequently, we focused on the development of

bio-resorbable membrane prepared from chitosan and CaSO4,

and attempted to evaluate the feasibility of devising composite

membranes for GTR applications.

We previously reported that the chitosan membranes

prepared by thermally induced phase separation method

followed by treatment with non-toxic NaOH-gelating,

Na5P3O10- and Na2SO3- cross-linking agents exhibited similar

properties with chitosan membrane that had been strengthened

by glutaraldehyde as a cross-linking agent [12,15,21]. In this

study, we took advantage of the unique properties of chitosan

and CaSO4 to prepare a composite membranes system. To

retain the good biocompatibility of these two unique materials

within the same device, we used NaOH as the gelating agent in

the process of preparing CaSO4-chitosan composite membrane.

SEM observation was conducted to examine the morphology of

the membrane surface. Also, some fundamental properties of

the CaSO4-chitosan composite membranes were examined,

such as water content, mechanical strength and degradation. In

addition to assess the physical properties of CaSO4-chitosan

composite membranes, the biological function of the composite

membranes was studied by animal test. We wish to establish

the feasibility of composite membranes made of the two

naturally occurring biomaterials, CaSO4 and chitosan, for GTR

in periodontal application.

2. Materials and methods

Chitosan with molecular weight of 300,000 and

deacetylation degree of 83% was purchased from TCI

(Tokyo, Japan). Chitosan with molecular weight of 70,000

and deacetylation degree of 75% was purchased from Sigma

(St. Louis, MO). CaSO4 (Merck-Schuchardt, Germany) was

classified through a sieve with 0.104 mm openings. Acetic

acid was purchased from Sigma (St. Louis, MO). All

chemicals used in this study were of reagent grade.

2.1 Preparation of CaSO4-chitosan composite membranes

Chitosan was dissolved in acetic acid (0.1M) to prepare a

2% (w/v) chitosan solution. This chitosan solution was filtered

and mixed with CaSO4, weight of ratio CaSO4/chitosan 65:35,

and then stirred 24 hours. Each 19 ml of mixed solution was

poured into a 9-cm Petri-dish and placed in a drying oven at

40°C with proper ventilation for overnight. After drying, the

membranes were immersed in 0.1N NaOH 4 hours, and then

washed with distilled water and pressed from two sides with

polyethylene (PE) thin films, and then placed in oven (40°C)

until completely dry. The chitosan-containing CaSO4 membranes

exhibited ivory-like uniform and opaque texture. All prepared

chitosan membranes were stored in desiccators until use.

2.2 Water content measurement

The water content (W.C.) of the membrane was

determined by swelling the membrane in pH7.4 of phosphate

buffered saline (PBS) at room temperature. After the membrane

reached equilibrium state, the wet membrane was blotted with

filter paper to remove the water adhered on the surface. The

water content of the membrane was calculated as:

. . ( ) / 100%w d wW C W W W= − × (1)

where Ww and Wd are the weights of wet and dry membrane,

respectively. The experiment was conducted 3 times and a

mean and standard deviation were calculated.

2.3 Mechanical property measurement

The mechanical properties of the membranes were

measured in hydrated condition. The membranes, 1 cm × 6 cm,

were hydrated in 0.1 M pH 7.4 phosphate buffer before being

subjected to mechanical testing. The tensile strength

measurements of the membranes were charted up to the point

where they were broken. The mechanical parameters of these

chitosan membranes were calculated and recorded

automatically by using an MTS Systems (Eden Prairie, USA) at

a crosshead speed of 10 mm/min.

2.4 In vitro degradation test

The in vitro degradation tests of the prepared membranes

were conducted by incubating the membrane in 10 ml of pH

7.4 PBS on a shaker set at 40 rpm and 37°C. At predetermined

time intervals, the membrane was taken out of the incubation

medium, washed with distilled water, dried and its weight was

measured. Another fresh 10 ml PBS was added into the vial for

continuum degradation test. The degradation profiles were

expressed as the accumulated weight losses of the membrane.

2.5 SEM observation

The surface microstructure of membranes was examined by

scanning electron microscope (SEM). Before SEM observation,

all samples were dried, sputter-coated with gold, and examined

under a scanning electron microscope (JEOL, JSM-5300, Japan).

GTR with use of CaSO4-Chitosan Membrane 306

Figure 1. (a) Dental round burr. (b) Macroscopic appearance of an experimental site. A bone defect (6 mm in diameter) was created at skull of

a rabbit. A defect on the skull covered with (c) chitosan (70 kDa) membrane, (d) chitosan (30 kDa) membrane,

(e) CaSO4-Chitosan (70 kDa) composite membrane, and (f) CaSO4-Chitosan (300 kDa) composite membrane (10×10 mm2 in area).

Figure 2. SEM micrographs of the membrane: (a-b) a chitosan (70 kDa) membrane, (c-d) a chitosan (300 kDa) membrane membrane, (e-f)

CaSO4-Chitosan (70 kDa) composite membrane, and (g-h) CaSO4-Chitosan (300 kDa) composite membrane.

2.6 Animals and operation procedure

SD rats weighing about 200 g, fed with commercial

food and RO water, were included in this randomized,

blinded study. The rats were anesthetized by

trans-abdominal injection of Zoletil 50 (mixture of

Tiletamine and Zolezepam, 1:1) (0.2 ml/200 g). Following

the injection, the skull was shaved and the surfaces at

surrounding sides of the skull were exposed via

full-thickness incision. A man-made defect (6 mm in

diameter) was generated with a dental round burr (Figure

1(a)). Prior to implantation, the membranes were hydrated

in physiologic saline to restore their elasticity. The defect

was then covered with the membranes (Figures 1(b-e),

10 × 10 mm2 in area, 0.1 mm in thickness). In the control

group, the bone defect was not covered with any chitosan

membrane. The wounds were then carefully sutured. For

each animal with membrane and control, an initial healing

period of 4 weeks was allowed.

2.7 Histological preparation and evaluation

After the healing periods, the rats were sacrificed by

injecting an overdose of KCl into the heart. The skull tissue

containing bone defects was removed by a larger-size dental

trephine burr. The specimens of center of defect were fixed in

10% neutral-buffered formalin, decalcified in 10% formic acid

and then dehydrated in an ascending graded series of ethanol

solutions, and afterwards, embedded in paraffin. A series of

5-µm transverse sections encompassing the entire bone defect

specimen were prepared and stained with hematoxylin-eosin

and then subjected to light microscopic observation.

3. Results

3.1 Morphology of CaSO4-chitosan composite membrane

As indicated in Figure 2, the SEM micrographs showed

the characteristic morphological aspects of various chitosan

membranes. All chitosan membranes produced by thermally

J. Med. Biol. Eng., Vol. 29. No. 6 2009 307

(a)

(b)

Figure 3. EDX analysis of (a) CaSO4-Chitosan (70 kDa) and (b) CaSO4-Chitosan (300 kDa) composite membrane.

Table 1. Physical Properties of CaSO4/Chitosan CompositeMembranes (Average ± SD, n = 4).

Equilibrium water

content at 24 hr (%)

Young’s modulus

(MPa*)

Ultimate elongation

(mm)

Ultimate strength

(Pa)

CaSO4/70 kDa Chitosan 36.0 ± 2.4 24.7 8.0 ± 0.8 190.7 ± 11.7

CaSO4/300 kDa Chitosan 46.2 ± 0.7 18.8 21.6 ± 4.1 385.3 ± 63.0

Pure 300 kDa Chitosan 53.1 ± 1.1 15.0 10.9 ± 3.2 169.3 ± 64.7

*Chitosan membranes were hydrated in pH 7.4 PBS before experiment run.

induced phase separation demonstrated a dense

morphological structure. The roughness was visible on the

CaSO4-chitosan composite membranes. Contrarily, the pure

chitosan membrane showed a smoother surface as compared

with all CaSO4-chitosan composite membranes. Furthermore,

with elemental analysis using EDX (Figure 3), calcium could

be detected on the surfaces of the composite membranes.

Interestingly, the calcium ratio of the CaSO4-chitosan

(300 kDa) composite membrane surface was higher than that

of CaSO4-chitosan (70 kDa). It manifested that CaSO4 could

be “float” more on the surface of the membrane when using

300 kDa chitosan as the source of binding material.

3.2 Basic properties of CaSO4-chitosan composite membrane

When considering the development of guided tissue

regeneration barrier materials, the basic bulk and mechanical

properties ought to be reviewed. In addition, the appropriate

degradation rate of a material has to be evaluated to assess

whether it meets the requirements and diversities of tissue

regeneration procedures. Interestingly, CaSO4-chitosan

composite membranes have become very desirable elastomers

to biomedical engineers, as indicated by the wider range of

physical characteristics, especially with higher ultimate length.

As expected, the presence of CaSO4 would affect Young’s

modulus of the membranes in this study, as shown in Table 1.

The Young’s modulus of the membrane increased from 15.0

MPa (for pure chitosan) to 18.9 MPa CaSO4-chitosan

composite membrane.

Moreover, as shown in Figure 4, the CaSO4-chitosan

composite membranes showed a more rapid mass loss than the

pure chitosan membranes in 30-day shaking test. All

CaSO4-chitosan composite membranes degraded to about

30%–50% of initial weight after 30-day shaking test. For the

composite membranes, the degradation behavior showed a

typical biphasic profile. There was an initial rush of

degradation during the first 6 days of the study. This rapid

Mass

lo

ss (

%)

Time (days)

Figure 4. The degradation profile of the membranes in pH7.4 PBS

solution at 37°C for 30-day shaking.

degradation period probably represented the release of feebly

entrapped and surface-associated CaSO4. After the first 6

days, the composite membranes showed slower degradation

rates. However, the degradation rates were slightly different

between the composite membranes. The degradation of

CaSO4-chitosan composite membranes increased with the

decrease of the molecular weight of chitosan, probably due

to CaSO4 on the CaSO4-chitosan (300 kDa) composite

membrane surface being richer than that of CaSO4-chitosan

(70 kDa). As a result, in the first 6 days of degradation, the

calcium ion was released faster from the membrane surface.

Overall speaking, the CaSO4-chitosan (300 kDa) composite

membranes showed a more rapid mass loss. In other words,

the degradation result corresponded roughly to the EDX

analytical result.

Figure 5 shows the surface morphologies of the

CaSO4-chitosan composite membranes after 30 days of

sh ak in g . SEM examin a t ion s r evea l ed th at th ese

CaSO4-chitosan composite membranes still exhibited rough

GTR with use of CaSO4-Chitosan Membrane 308

Figure 5. SEM micrographs of the membrane: (a-b) a chitosan (70 kDa) membrane, (c-d) a chitosan (300 kDa) membrane membrane, (e-f)

CaSO4-Chitosan (70 kDa) composite membrane, and (g-h) CaSO4-Chitosan (300 kDa) composite membrane after 30 days of

shaking.

Mass

lo

ss (

%)

Time (days)

Figure 6. Water contact profile of the membranes in pH 7.4 PBS

solution in 10-minute period of experiment.

surfaces and had good integrity, albeit with signs of

degradation on the surface. CaSO4 was steadily released

and dissolved from the membranes while, concurrently,

chitosan was degraded only gradually. Relatively speaking,

the surface of neat chitosan retained an intact and smooth

surface morphology as before. As a guided regeneration

barrier material, the appropriate degradation rate of

material ought to be managed to fit into the schedule of

remodeling of tissue regeneration. Although, the resorption

process could be further facilitated by enzyme digestion in

real applications, we suggested that the membranes

prepared in this study could reasonably meet the

degradation requirement of bioresorbable membrane used

for GTR from these in vitro observations.

Another important characteristic of these chitosan

membranes is that they could reach steady hydration

equilibrium state within the 10-minute period of the

experiment, as seen in Figure 6 showing water content

profiles. This rapid swelling phenomenon would be

beneficial to the clinical manageability and surgical

procedures. The bulk properties of prepared

CaSO4-chitosan composite membrane are summarized in

Table 1. The results indicated that the CaSO4-chitosan

composite membranes prepared in this study fulfilled the

requirements of bioresorbable membrane employed as GTR

material. In clinical practice, when materials are used as

tissue regeneration barrier membranes, they are generally

required to maintain certain barrier functions for 4 to 6

weeks in order to secure the successful restoration of

periodontal tissues. The mechanical strength test results

indicated that these membranes had appropriate mechanical

strength for the requirement of GTR.

3.3 Histological observations

Varying degrees of bone healing were observed beneath

the CaSO4-chitosan composite membranes in comparison to

the control group. Figure 7 shows a typical transverse section

of experimental bone defect 4 weeks after surgery. In the

control group, the connective tissue grew into the bone

defect area and prevented the bony cells from growing back

to their natural form or space and thus destroyed the healthy

process of new bone growth (Figure 7(a)). Interestingly, in

the experimental groups (i.e., defect covered with

CaSO4-chitosan (300 kDa), and CaSO4-chitosan (70 kDa)

composite membranes), the connective tissue cells had only

limited proliferation on their original sites and prevented the

connective tissue cells from intruding into the space of the

bone defect (Figures 7(b)-(e)). Sequentially speaking, the

bone defect was allowed with the space and critical healing

time to be repaired by newly yet slowly formed bone that

proliferated in regions partially or fully protected and

separated by the CaSO4-chitosan composite membrane. This

process practically prevented any connective tissue from

invading into the bone defect. Furthermore, no obvious

inflammatory response was observed around the chitosan

membrane at this initial healing stage. However, the bone

healing seemed to be slowed down while the chitosan

membrane still occupied and saved the space exclusively for

the bone tissue to be healed in this isolated area (as compared

to the control).

J. Med. Biol. Eng., Vol. 29. No. 6 2009 309

(a)

(b)

(c)

(d)

(e)

Figure 7. Histological section of skull portion of a rat undergoing GTR procedure 4 weeks after surgery; (a) control, without chitosan

membrane coverage, (b) with chitosan (70 kDa) membrane coverage, (c) with chitosan (300 kDa) membrane coverage,(d)

CaSO4-Chitosan (70 kDa) composite membrane coverage, and (e) with CaSO4-Chitosan (300 kDa) composite membrane coverage.

Hematoxylin and eosin stain; original magnification × 100.

The preliminary results demonstrated that these prepared

CaSO4-chitosan composite membranes in this study could

successfully isolate the bone defect from ingress of connective

tissue cells and provide a space where bony tissue cells could

grow into later. There are other advantages, including the

readily gel-forming properties, and being easily available in

pure form, biodegradable and osteoinductive. Full utilization

of the characteristics of chitosan could allow it to become a

promising material in GTR applications.

4. Discussion

Many aspects of technical development of bioresorbable

membrane materials for GTR applications are focusing on

their rigidity and degradation rate, and with special emphasis

on the ease of clinical manageability. Lots of effort has been

spent on improving the biological function of barrier

membrane. For example, surfaces coated with alginate were

found to resist cell adhesion. In this study, we brought together

two attractive biomaterials, CaSO4 and chitosan, which are in

abundance and inexpensive, and possess excellent

biocompatibility and biodegradability, in applications for GTR.

Consequently, we have presented here a simple phase-induced

separation method to prepare a series of CaSO4-chitosan

barrier membranes. Briefly, CaSO4 was annexed to chitosan

matrix to prepare the composite membranes with high

mechanical strength. Following the process, CaSO4 also

altered morphological structure and degradation behavior of

the chitosan membranes. These changes probably are

attributed the ability of CaSO4 to bind with chitosan through

ionic bonding, and subsequently, to cross-link to the different

parts of chitosan polymer chains. To verify this postulation, we

tested the membranes in a medium of 0.1 N acetic acid. We

observed that CaSO4-chitosan membranes were not dissolved

completely even after 24-hour of shaking (not shown),

whereas a neat chitosan membrane would be readily dissolved.

Subsequently, we applied these CaSO4-chitosan

composite membranes to the animal GTR study models.

GTR with use of CaSO4-Chitosan Membrane 310

Based on the findings from the histological evaluation, all

three prepared chitosan membranes apparently exhibited

better membrane integrity in bone defect healing after 4

weeks and provided good cell separation ability (see Figures

7(b)-7(e)) compared with controls (Figure 7(a)). Among the

chitosan membranes tested, the defects covered with

CaSO4-chitosan composite membranes had higher percentage

of new bone formation. On the contrary, in the case of the

control group, the connective tissue might have grown

preemptively into the bone defect area, and that caused the

bony cells, with much slower growth rate, to not be able to

grow back into their originally designated space and form.

Another important factor for the success of GTR techniques

is that the barrier material ought to withstand a period long

enough for the bony tissue to reach sufficient healing stages.

In clinical practice, the tissue regeneration barrier

membranes are generally required to maintain their barrier

functions for 4 to 6 weeks, with ample mechanical strength

left, in order to secure the restoration of periodontal tissues.

As observed from Figure 5, it could be concluded that these

chitosan membranes were apparently suited for GTR in

biodegradable characteristics.

5. Conclusion

On the basis of the results observed, it could be

concluded that the CaSO4-chitosan composite membranes

prepared in this study appeared to be of great promise for

application in GTR in general. However, to assess and

explore the full potential of these materials in physiologically

more demanding periodontal applications for GTR, we shall

need to perform extensive and in-depth studies of animal

models that more closely resemble to the human anatomy,

such as in the porcine oral cavities.

Acknowledgements

This work was supported by grants from the National

Science Council, Taiwan (94-2213-E-214-038, 96-2815-C-214

-018-E).

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