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Osteogenic response to porous hydroxyapatite ceramics under the skin of dogs

Hiroshi Yamasaki Department of Dentistry and Oral Surgery, School of Medicine, Fukuoka University, 7-45-l Nanakuma, Johnan-ku, Fukuoka, 814-01, Japan

Hidetaka Sakai Department of Oral Pathology, Faculty of Dentistry, Kyushu University, 3-l-l Maidashi, Higashi-ku, Fukuoka, 872, Japan

Soft tissue reactions to two materials of different microstructure made with porous and dense hydroxyapatite ceramic granules, were compared in this study. Each was implanted subcutaneously in the abdomen of 10 dogs. The implants were removed 1, 3 and 6 months after implantation, and examined by light microscopy. Heterotopic bone formation was only evident around porous hydroxyapatite ceramic granules in 6 of the 10 animals after 3 months, and in all cases after 6 months, but never around dense granules. These findings suggested that cellular differentiation and induction of osteogenesis may be influenced by the physical and chemical factors in the micromilieu provided by porous hydroxyapatite ceramic granules.

Keywords: Hydroxyapatite, microstructure osteogenesis, tissue reaction

Received 12 November 1990; revised 24 January 1991; accepted 23 March 1991

Hydroxyapatite (HA) ceramics implanted in osseous sites bind directly with bone. This material has been applied clinically as a bone substitute’-3. Bone formation around an implanted HA ceramic substitute is generally thought to occur by osteoconduction from surrounding osseous tissues4. However, the extent of bone formation within the structure of porous HA raises the question of whether porous HA acts as a scaffold for bony ingrowth and has a passive role in the induction of bone, or whether it acts as an osteoinductive material’.

In non-osseous sites, on the other hand, no report has been made that HA ceramics promote bone formation. Most investigators believe that this biomaterial is devoid of intrinsic osteoinductive properties’-‘.

Recently Heughebaert et aI.” reported that the physico-chemical analyses of porous HA ceramics implanted in hamster soft tissues for 12 months, demonstrated the formation of bone-like material.

The purpose of this study was to evaluate the soft tissue response to the subcutaneous placement of porous and dense HA ceramic granules, and specifically to search for any histological evidence of bone formation around them.

Correspondence to Dr H. Yamasaki.

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MATERIALS AND METHODS

Materials

Two types of HA ceramic granules, porous and dense, were wet synthesized and sintered at 1200’C (Asahi Optical Co., Tokyo, Japan]. These granules ranged in size from 200 to 600 pm and porous granules had a continuous and interconnected microporosity ranging in diameter from 2 to 10 pm (Figure 2). The X-ray diffraction analysis showed that these granules were almost pure hydroxy- apatite (>99.9%) without tricalcium phosphate and pirophosphate (Figure 2). The infrared absorption spectroscopy data of these granules showed that the ceramics were not decomposed and there were hydroxyl groups.

Methods

Ten adult male mongrel dogs anaesthetized with sodium pentobarbital were used in all experiments. The abdomen of each dog was shaved and prepared with a povidone- iodine solution, and six separate subcutaneous pockets were created by blunt dissection. Porous granules were implanted on the left and dense granules were implanted

0 1992 Butterworth-Heinemann Ltd 0142-9612/92/050308-05

Osteogenic response to hydroxyapatites: H. Yamasaki and H. Sakai 309

Figure 1 Scanning electron photomicrograph of the surface of: a, porous, b, dense hydroxyapatite ceramic granules.

c)

5 s

L L 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40

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Figure 2 X-ray diffraction pattern of porous hydroxyapatite (HA) ceramic granules, showing that the granules are >99.9% pure hydroxyapatite. That of dense type shows same findings.

on the right side, both in sites far from osseous tissues, The implant quantities were -1 g each. The animals were administered antibiotic prophylaxis orally for 4 d after the implantation procedure.

The implants were removed along with the surrounding tissue 1,3 and 6 months after implantation. All specimens were fixed in neutrally buffered formalin, decalcified using the Plank-Rychlo technique, and examined by light microscopy.

Figure 3 The hydroxyapatite (HA) mass of the porous-type granules within the subcutis of dog abdomen is shown to be mobile and has a bone-like consistency 3 months after implantation.

RESULTS

Macroscopic observations

Each implanted HA mass of granules was mobile when palpated, and many of the porous type had a bone-like consistency, 3 and 6 months after implantation (Figure 3).

Microscopic observations

Neither porous or dense HA ceramics showed bone formation histologically, 1 month after implantation. These granules were surrounded with fibrous connective tissue and partially multinucleated giant cells appeared in the specimens. Three months after implantation, newly formed bone tissue was revealed around the porous HA ceramics in six of ten animals studied, but no ossification was found at any implantation site of the dense HA ceramic. The HA mass was localized within the subcutis and surrounded with fibrous connective tissue (Figure 4).

The newly-formed bone tissue presented features of intramembranous ossification without cartilage for- mation. The new osseous tissue was primarily immature trabecular bone with intralacunar osteocytes, with obvious osteoblastic rimming [Figure 5). The osteoblasts were seen near the surface of the porous HA ceramics, along with adjacent bone-matrix formation and pro- liferation of preosteoblast-like cells with large nuclei [Figure 6). The micropores of the HA ceramics were found to be filled with eosinophilic amorphous sub- stance, suggesting a bone matrix. The dense HA ceramics were surrounded by collagen fibres and occasional macrophages and multinucleated giant cells, but in no case was there evidence of bone-matrix formation or calcification at 3 months [Figure 7).

Six months after implantation, the specimens from the porous type revealed new bone formation in all animals, and fatty bone marrows were seen among the bone trabeculae in four of ten dogs [Figure 8). However, neither ossification or calcification was found in the specimens from the dense type, and, on the surface of HA ceramics, macrophages and multinucleated giant cells were prominent [Figure 9).

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310 Osteogenic response to hvdroxyapatites: H. Yamasaki and H. Sakai

Figure 4 Specimens, 3 months after implantation, show the porous hydroxyapatite (HA) ceramic granules surrounded with fibrous connective tissue within the subcutis. The arrow in a, indicates the region highlighted in b. New bone formation around the hydroxyapatite (HA) ceramic granules and trabe- cular structures are demonstrated. P: porous hydroxyapatite (HA) ceramics (original magnification: a, X6; b, X25).

Figure5 Higher-power views of the new bone around the porous hydroxyapatite (HA) ceramics. Osteoblastic rimming around the new bone and intralacunar osteocytes can be noted (original magnification X100).

DISCUSSION

Clinically, heterotopic bone formation is encountered as a complication in spinal cord injury and hemiplegia. Experiments have demonstrated heterotopic bone for-

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Figure 6 Differentiating osteoblasts and bone-matrix for- mation are seen near the porous hydroxyapatite (HA) ceramics. Small arrows in a and b show eosinophilic substance within the micropore of the porous hydroxyapatite (HA) ceramic granules. Large arrow in a shows an preosteoblast-like cell. P: porous hydroxyapatite (HA) ceramics (original magnification: a, x100, b, x50).

D

Figure 7 Dense hydroxyapatite (HA) ceramics are surrounded by collagen fibres. No osseous tissue is found 3 months after implantation. D: dense hydroxyapatite (HA) ceramics (original magnification X100).

mation in animals bearing grafts of urinary tract tissues” and in those with implanted poly(hydroxyethy1 meth- acrylate) sponge . I2 Heterotopic bone formation has also been seen in animals with bone morphogenetic protein

Osteogenic response to hydroxyapatites: H. Yamasaki and H. Sakai 311

Figure8 Specimen from the porous hydroxyapatite (HA) ceramic, 6 months after implantation, showing new bone formation and fatty bone marrow among the bone trabeculae. P: porous hydroxyapatite (HA) ceramics, M: bone marrow (original magnification X25).

Figure 9 Macrophages and multinucleated giant cells are prominent on the surface of the de;ise hydroxyapatite (HA) ceramics 6 months after implantation. D: dense hydroxyapatite (HA) ceramics (original magnification X100).

(BMP) extracted from bone matrix, but the underlying cause and mechanism remain unclear4.

Experiments on HA ceramic implantation in soft tissues have been reported, and some have involved long- term observations, although none presented evidence of osteogenesis. Most of these studies dealt with dense HA ceramics’3-‘6, whilst studies of porous HA ceramics comprised short-term observations of less than 3 months5+‘.

Heughebaert et aI.” reported that the physico- chemical analyses of porous HA ceramic implanted in soft tissues of hamsters for 12 months demonstrated the formation of bone-like material and they thought the HA ceramic promoted new bone formation (osteoinduction). However, they also reported that the new material did not give the histological and morphological character- istics of true bone.

Recently we found that the subcutaneous implan- tation of porous HA ceramic granules in the abdomen of the dog, resulted in heterotopic bone formation, 3 months after implantation17.

However, many reports have appeared concerning experimental implantation of HA ceramics in or on bone’8-24. Some of the reports describe the observation of newly-formed osseous tissue, as well as the differentiation of osteoblasts at the surface of the HA ceramic implant. These reactions have generally been interpreted as a process of extension of bone growth from the surrounding osseous tissues’8’ 24. However, some investigators consider them to be a sign of the osteoinductive processes’” “, and the extent of bone formation within the structure of porous HA has raised the question of whether porous HA acts as a scaffold for bony ingrowth and has a passive role in the induction of bone, or whether it acts as an osteoinductive materia15.

Reddi and Hugginsz5 reported that osteogenic trans- formation of fibroblasts was profoundly influenced by the geometry of the transformants, such as demineralized bone powder and whole teeth.

El Deeb et aL5 suggested that physical factors, including the three-dimensional configuration of porous HA ceramics, were important in bone formation within the material.

In this study, osteoblast differentiation and new bone formation at the surface of implanted porous HA ceramic granules were obviously demonstrated in non- osseous sites. The micropores of the HA ceramics were filled with eosinophilic amorphous substance, suggesting the ingrowth of connective tissue9 or unknown organic materials’“. On the contrary, there was no evidence of newly-formed osseous tissue at the implantation site of dense HA ceramic granules.

These findings suggest that physical and chemical factors in the micromilieu provided by implanted HA ceramic granules possessing micropores exert an effect on osteogenesis.

We speculate that they may be due to calcium phosphates leaching out of the porous granules or the ion-exchange reactions in the adjacent tissue. The release of co-phosphate can depend not only on the porosity of HA samples, but also on other accompanying amorphous material not detectable from X-ray analyses or infrared spectroscopy.

ACKNOWLEDGEMENT

The authors thank Professor H. Miyako for supporting this study. M. Kishimoto for technical assistance, and Y. Hirayama (Asahi Optical Co., Tokyo, Japan) for preparing the implant materials used in this study.

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