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Experimental studies on hydroxyapatite powder-carboxymethyl chitin composite: injectable material for bone augmentation
Journal of Plastic, Reconstructive & Aesthetic Surgery (2006) 59, 188–196
Experimental studies on hydroxyapatite powdercarboxymethyl chitin composite: injectable material for bone augmentation Hirokazu Udaa,*, Yasushi Sugawaraa, Masayoshi Nakasub a
Department of Plastic and Reconstructive Surgery, Jichi Medical School, Tochigi, Japan New Ceramics Department, Asahi Optical Co. Ltd, Tokyo, Japan
b
Received 11 January 2002; accepted 30 November 2004
KEYWORDS Hydroxyapatite; Carboxymethyl chitin; Bone augmentation; Physical gels
Summary We developed a hydroxyapatite (HA) powder-carboxymethyl chitin composite (HA-CMC composite) that can be injected with a 14G needle by adding distilled water. We prepared Materials I (HAZ57.0 wt%) and II (HAZ40.2 wt%) and examined their biocompatibility and osteoconductivity. With a 2-mm skin stab, the material was injected on the calvarial bone of rats. The periosteum was denuded blindly in half of the cases and preserved in the other half of the cases. Simultaneously, the material was injected subdermally into the abdominal skin to examine diachronic volume alteration of the material. Our results indicated that the new materials had biocompatibility as high as that achieved with previously developed HA materials. The difference in HA concentration did not influence the osteoconductivity, but the periosteum and the soft tissue on the cranium seemed to be an obstacle to bone ingrowth. On the other hand, the volume alteration was significantly smaller in Material I than in Material II. This composite may be especially useful in facial bone augmentation because it can be injected with only a small skin stab. When used for that purpose, the periosteum of the host bone should be denuded to facilitate bone ingrowth, and Material I will be preferable to Material II in terms of the maintenance of the initial volume. Q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
Bone substitute must be easy to shape, easy to handle, and able to maintain its initial volume. Hydroxyapatite (HA), as is now well known, has high biocompatibility and osteoconductivity, and it has
* Corresponding author. E-mail address: [email protected] (H. Uda).
been widely used as a bone substitute material in orthopaedic, periodontal, maxillofacial, and reconstructive surgery.1–4 However, some difficulties have been reported. HA blocks are usually difficult to shape to the desired form and troublesome to fit to the recipient side intraoperatively.4–6 HA granules can be applied to an uneven surface of a
S0007-1226/$ - see front matter Q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2004.11.022
Experimental studies on hydroxyapatite carboxymethyl chitin composite recipient bone, but are difficult to handle and may not retain their initial shape and volume because of secondary migration. 4,7,8 Calcium phosphate cement has been used in an attempt to overcome these disadvantages,7,9–11 but the effects of blood and tissue fluid at the recipient site prevents this material from transforming HA, leading to secondary migration and partial absorption.12 Thus, even calcium phosphate cement requires a dry field at the recipient site, and a sizeable skin incision is needed to achieve sufficient hemostasis.13 This skin incision limits the clinical use of HA, especially in facial bone augmentation. To address that problem, we have developed a hydroxyapatite powdercarboxymethyl chitin composite (HA-CMC composite). This system is dry and epispastic generally and can be gelled and injected with a 14G needle by adding distilled water (Fig. 1). In this system, the degradable carboxymethyl chitin (CM chitin) acts as an injectable material and binder to prevent the migration of HA grains from the recipient site. In this report, we described the preliminary development of HA-CMC composite, our observation of its biocompatibility histologically, and the results of our quantitative examination of its osteoconductivity by means of histomorphometry, as well as the rate of volume alteration after implantation by experiments on rats.
Materials and methods HA powder and CM chitin were obtained from Asahi Optical Co. (Tokyo, Japan). The diameter of the HA grain ranged from 100 to 300 mm, and the shape of the grain was inconstant (Fig. 2).
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Figure 2 Electron micrograph of HA grains. The diameter of the HA grain ranged from 100 to 300 mm, and the shape of the grain was inconstant.
equal in the two materials as 2.0 percent weight by adding the proper amount of distilled water. By using two connected syringes, one filled with distilled water and another filled with HA-CMC composite, it was easy to achieve homogeneous distribution of the HA in the chitin gel (Fig. 3), and the homogeneous distribution could be maintained due to the viscosity of the chitin gel.
Implantation procedures We used female Wister rats, 6 to 8 weeks old. After mixing the HA-CMC composites and distilled water using a 5-ml syringe, we made a subdermal pocket in the head of each rat with a small 2-mm skin stab. The composite material was then injected with a 14G needle on the calvarial bone Fig. 4. At that time the periosteum was denuded blindly through the
Preparation of HA-CMC composite We prepared two kinds of HA-CMC composites, Materials I and II. Table 1 shows the amount of reagent in the preparation. The proportion of HA in Material I was higher than that in Material II (Material IZ57.0 wt%, Material IIZ40.2 wt%), and the concentrations of CM chitin were adjusted to be
Figure 1 The test material can be gelled and injected easily with a 14G needle by adding distilled water.
Figure 3 By using two connected syringes, one filled with distilled water and another filled with HA-CMC composites (a), it was easy to achieve homogeneous distribution of the HA in the chitin gel (b).
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Table 1
Material I Material II a
Preparation of HA-CMC composite in one vial HA (g)
CM chitin (g)a
Distilled water (ml)
Weight ratio of HA (wt%)
2.72 1.41
0.04 0.04
2 2
57.0 40.2
Adding 2 ml distilled water, the concentration of CM chitin solution was 2 wt% in each material.
skin stab with a small dissector in half of the cases, and it was preserved in the rest of the cases (Fig. 5). In addition, to examine diachronic volume alteration of the materials, we simultaneously injected 1 ml of the same material subdermally into the abdominal skin. Rats receiving these procedures were divided into the following groups (Table 2): Material I and periosteum was denuded (nZ16, group A), Material I and periosteum was preserved (nZ16, group B), Material II and periosteum was denuded (nZ16, group C), and Material II and periosteum was preserved (nZ16, group D).
Histological evaluation In all groups, the rats were sacrificed 2, 4, 8, or 16 weeks after implantation, respectively, (nZ4 at each time period). After macroscopic observation, implanted materials on the heads of rats were delivered en bloc with calvarial bones. The samples were placed immediately into 10% buffered formalin solution and examined histologically.
Histologic specimens were decalcified, embedded in paraffin, and cut into 4.5 mm thick sections just on the centre of the posterior frontal suture along the long axis of calvarial bones. The specimens were stained with hematoxylin and eosin and then examined with a light microscope. Next, they were diachronically examined for soft tissue reaction around the HA granules such as inflammation and its maturation. We also determined the bone ingrowth rates of all four groups by quantitative histomorphometric examination of osteoconductivity in all groups. With the histological sections at the sagittal plane just on the centre of the posterior frontal suture Fig. 6, of which the length was 3 mm and the height was 1.5 mm, we determined the areas of new bone ingrowth and calculated the bone ingrowth rates {(the area of new bone ingrowth/3!1.5 mm2)! 100(%)}. These were analysed using Adobe Photoshop 7.0 and NIH Image 1.63.
Evaluation of the rate of materials volume alteration In all groups, the materials injected subdermally into the abdominal skin were harvested simultaneously when the rats were sacrificed, and in each cases the volume of the materials was immediately measured with a volumeter to examine volume alteration of the materials. We examined the diachronic rates of volume alteration in Materials I and II.
Figure 4 A subdermal pocket was made in the head of each rat (patched area) with a small 2-mm skin stab. The composite material was then injected with a 14G needle on the calvarial bone.
Figure 5 During the injection of materials, the periosteum was preserved in half of the cases (a) and denuded blindly through the skin stab with a small dissector in the rest of the cases (b).
Experimental studies on hydroxyapatite carboxymethyl chitin composite Table 2
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Dividing groups
Group
A
B
C
D
Material Periosteum
I Torn off
I Preserved
II Torn off
II Preserved
The cases were divided into four groups. nZ16 in all.
Results Gross findings Even at 2 weeks after implantation, the head and nasal dorsum in which the material was injected were well augmented. When we dissected the skin and observed the material directly, the material was already covered entirely with a thin, pellucid capsule and the material had retained its initial shape. No secondary migration of HA grains was seen. These findings could be seen in all cases of groups A–D.
Histological findings In all groups, all interspaces between HA grains were filled with immature granulation tissue at 2 weeks after implantation, and each grain was surrounded by this tissue. We observed a slight infiltration of inflammatory cells. Simultaneously, some foreign body giant cells stuck to HA grains were defined (Fig. 7(a) and (b)). The granulation tissue, mainly composed by fibroblast, had been matured diachronically, and each HA grain was well fixed with mature collagen fibre and kept from migrating at 16 weeks. Foreign body giant cells still existed, but no englobement of HA grains was seen (Fig. 7(c) and (d)) (Figs. 7 and 8). In groups A and C, in which the periosteum was denuded blindly, we found immature granulation tissue in most of the contact area between the
material and the calvarial bone; we also saw a little new bone formation. However, at 4 weeks after implantation, there was additional new bone formation, and it had spread along the material surface facing the host bone (Fig. 8(a) and (b)). New bone seemed to increase with time. At 16 weeks after implantation, almost all contact surface of the material was bound with new bone, and partially deep ingrowth of the new bone was observed (Fig. 8(c) and (d)). Regarding the soft tissue response to the materials, it seemed that there were no apparent difference among groups A, B, C, and D. With respect to the hard tissue response to the materials, no bone ingrowth was seen in any cases of groups B and D, in which the periosteum was preserved, throughout all the test periods. Even 16 weeks after implantation, coarse connective tissue seemed to prevent new bone ingrowth to the material (Fig. 8(e)). In contrast, quantitative examination of osteoconductivity revealed that the bone ingrowth rate gained diachronically in the cases of groups A and C, in which the periosteum was denuded. Statistical analysis (using the Mann–Whitney U tests and assuming unequal variances, we compared the bone ingrowth rates at 2, 4, 8, and 16 weeks after implantation; statistical significance was defined as P!0.05) revealed there were no significant differences in the bone ingrowth rate between the cases of groups A and C at 2, 4, 8, and 16 weeks after implantation (Fig. 9). That is, the osteoconductivity did not depend on Materials I and II.
Volume alteration of the materials
Figure 6 The areas of new bone ingrowth were calculated with the histological sections, of which the length was 3 mm and the height was 1.5 mm just to the centre of the posterior frontal suture (PF: posterior frontal suture, Cor: coronal suture, Sag: sagittal suture, Lam: lamboid suture).
The volume of each material decreased uniformly at 2 weeks after implantation (Material I: meanZ 79%, Material II: meanZ63%). From 2 to 16 weeks after implantation, no significant volume alteration occurred in either material. As a whole, the volume of Material I was better retained than that of Material II. Statistical analysis (using the Mann– Whitney U test and assuming unequal variances, we compared the volume alterations of Materials I and II at 2, 4, 8, and 16 weeks after implantation; statistical significance was defined as P!0.05) between Materials I and II revealed that there
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Figure 7 Soft tissue response to the material in the case of group C (arrow: foreign body giant cell, *: HA grain). (a) and (b) At 2 weeks after implantation, the HA grains were surrounded by immature granulation tissues. A slight infiltration by inflammatory cells was observed, and some foreign body giant cells stuck to HA grain were defined (a: original magnification!40, b: original magnification!200). (c) and (d) At 16 weeks after implantation, the HA grains were well fixed with mature collagen fibre. Foreign body giant cells were still present, but englobement of HA grains was not seen (c: original magnification!40, d: original magnification!200).
were significant differences in volume alteration at 4, 8, and 16 weeks after implantation (Fig. 10).
Discussions HA has been widely used as a bone substitute material because of its high biocompatibility and osteoconductivity. However, for reconstructive and plastic surgeons who deal with facial bone, the originally developed HA materials are not a satisfying bone substitute, because in spite of recently developed calcium phosphate cement, a sizeable skin incision is needed for use of the material. This inevitable skin incision is a nuisance for facial bone augmentation. Our aim was to develop a facial bone augmentation material that can be injected without a sizeable skin incision. In our new material, HACMC composite, CM chitin acts to bind HA grains.
Previously, polymer-HA grain blends using binders such as collagen,14 alginate,15 polyhydroxybutyrate,16 fibrin glue,8 and chitosan17,18 had been reported, but these materials had various disadvantages. Some of them caused allergic reaction, some could not maintain homogeneity, some were excessively costly, some were difficult to preserve, some caused inflammation, and so on. As a result, these materials are not yet available clinically. In contrast, CM chitin, which is a derivative of chitin, may be suitable as a binder that can be used clinically. First, CM chitin is very economical, because chitin is one of the most abundant polymers in the biomass after cellulose and it is contained in crustaceans on a massive scale.19 Second, CM chitin is dry and epipastic in normal temperatures, so it is easy to preserve and easy to blend with HA grains. And last, CM chitin can be gelled easily by adding only distilled water, and it
Experimental studies on hydroxyapatite carboxymethyl chitin composite
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Figure 8 Hard tissue response to the material at the contact area between the material and the calvarial bone. Hematoxylin and eosin stain (*: HA grain, **: calvarial bone, arrow: new bone migration, arrowhead: periosteum). (a) and (b) At 4 weeks after implantation, in the case of group A, new bone spreading along the material surface facing the host bone was observed (a: original magnification!40, b: original magnification!100). (c) and (d) At 16 weeks after implantation, in the case of group A, almost all contact surface of the material was bound with new bone, and partially deep ingrowth of the new bone was observed (c: original magnification!40, d: original magnification!100). (e) At 16 weeks after implantation, in the case of group B, in which the periosteum was preserved, no new bone formation was seen even 16 weeks after implantation. Coarse connective tissue and the periosteum seemed to prevent new bone formation and its ingrowth to the material (original magnification!40).
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Figure 9 Quantitative examination of osteoconductivity revealed the bone ingrowth rate gained diachronically in the cases of groups A and C, in which the periosteum was denuded. Statistical analysis revealed there were no significant differences between the cases of groups A and C in the bone ingrowth rate at 2, 4, 8, and 16 weeks after implantation (using the Mann–Whitney U test and assuming unequal variances, we compared the bone ingrowth rates at 2, 4, 8, and 16 weeks after implantation; statistical significance was defined as P!0.05).
Figure 10 Statistical analysis (using the Mann–Whitney U test and assuming unequal variances, we compared the volume alterations of Materials I and II at 2, 4, 8, and 16 weeks after implantation) between Materials I and II revealed there were significant differences in volume alteration at 4, 8, and 16 weeks after implantation (*statistical significance was defined as P!0.05).
H. Uda et al. can maintain that property for a long time, unlike chitosan, which needs chemical modification with anhydrides or aldehydes to gelify.19 Moreover, the deacetylisation rate of our CM chitin is zero. Tokura and Tamura20 reported that the amino group in chitosan causes a bioactive reaction in the animal body, and in the same way the cytotoxicity of CM chitin is in proportion to the deacetylation rate of CM chitin. These differences indicate that our CM chitin material is superior to chitosan gel in regard to cytotoxicity. In this study, macroscopic observation showed satisfactory results of bone augmentation with the composite we developed. At 2 weeks after implantation, the material was entirely covered by a thin capsule and well fixed. Secondary migration of the material was not observed in any cases. At the same time, histological findings at two weeks after implantation revealed that each HA grain was enclosed and fixed by immature granulation tissue that seemed to prevent HA grains from migrating. These findings proved that CM chitin worked not only as an injectable binder of HA grains but also as a temporary scaffold. The granulation tissue matured gradually, and at 16 weeks after implantation it turned to mature collagen fibre, which in turn further fixed each HA grain. This finding proved that our system has the same high biocompatibility as that of previously developed HA substitutes. We found that the concentration of HA did not influence osteoconductivity in our material. In opposition to that, the results in groups A and C, in which the periosteum of the host bone was denuded blindly, were apparently superior to the results in groups B and D, in which the periosteum was preserved. We hypothesised that coarse connective tissue between the material and the host bone prevented new bone formation and bone ingrowth in the cases of which the periosteum was preserved. It should be noted that injury to the periosteum stimulates the cellular events that lead to passive bone ingrowth at the bone-implant interface. For bone augmentation, material should bond tightly to the recipient bone surface. We, therefore, believe the periosteum should be denuded when using this material clinically. As to volume decrease, we suspect that the early volume decrease of the material at 2 weeks after implantation (Material I: meanZ79%; Material II: meanZ63%) is due to absorption of water and CMchitin. After that, from 2 to 16 weeks, the volumes of both materials were well preserved. An ability to preserve initial volume is an invaluable property for bone augmentation material. In that respect, Material I was superior to Material II. The preservative capacity of the initial volume seemed to
Experimental studies on hydroxyapatite carboxymethyl chitin composite be in proportion to the HA rate of the composite. However, it was difficult to gain an HA rate higher than that of Material I, because injection with a 14G needle became impossible with higher rates. That problem may be solved by shrinking the HA grains or by changing the inconstant shape of the HA grains to a rounded shape. Additional resorption of material may occur in the long term, following the 16-week limit of our testing. In our results, histological findings at 16 weeks after implantation revealed that foreign body giant cells still existed around HA grains. Particles up to 15 mm can avoid phagocytosis;21 our HA grains were 100–300 mm to prevent englobement by phagocytes, but many minute fragments of HA grains smaller than 100 mm were actually observed. We supposed these fragments were mainly the result of physical demolition that occurred during when mixing of the composites and water with a syringe. These minute fragments might be digested in the long term, though englobement of HA grains was not seen in our histological sections at 16 weeks after implantation. Moreover, it has also been reported that phagolytic cells resorb calcium phosphate ceramics by two processes of dissolution and digestion.7,22,23 A much longer follow-up study may be needed to evaluate volume alteration of our material. Our material should be useful clinically, especially in cranio-maxillofacial surgery, as in, for example, revision of deformity after facial bone fracture and treatment for minor deformity of facial anomalies. Moreover, our system will be attractive in cases of cosmetic surgery, such as augmentation rhinoplasty, genioplasty, and malar bone augmentation, because only a small skin stab will be needed to administer the material. Based on the results of our study, we caution users that in those cases the periosteum of the host bone should be denuded by a small dissector to promote the osteoconductivity of the material, and the rate of HA grains should be higher (that is, Material I is superior to Material II). A few disadvantages became clear from our experience. First, we had difficulty forming this material into a detailed shape, because the shape depended on the shape of the subcutaneous pocket to some degree. Second, this composite is not selfhardening bone cement, so some period (about 5 to 7 days experimentally in this study) was necessary to fix the composite by fibrous capsule. And last, a certain decrease of initial volume, especially within the initial 2 weeks, occurred as mentioned above. There is room for improvement in our material. For instance, we would like to increase the rate of HA that can maintain its initial volume while also
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maintaining the material’s injectable property, and we would like to be able to prevent the generation of minute HA fragments due to physical demolition to protect against phagocytosis. However, we think this HA-CMC composite has good potential for expanded clinical use, especially for facial bone augmentation, because of its high biocompatibility, good osteoconductivity, easy handling, low cost, and easy preservation.
References 1. Parikh SN. Bone graft substitutes in modern orthopedics. Orthopedics 2002;25:1301–9 quiz 1310–1311. 2. Hes J, de Man K. Use of blocks of hydroxylapatite for secondary reconstruction of the orbital floor. Int J Oral Maxillofac Surg 1990;19:275–8. 3. Rosen HM, McFarland MM. The biologic behavior of hydroxyapatite implanted into the maxillofacial skeleton. Plast Reconstr Surg 1990;85:718–23. 4. Kent JN, Zide MF, Kay JF, Jarcho M. Hydroxylapatite blocks and particles as bone graft substitutes in orthognathic and reconstructive surgery. J Oral Maxillofac Surg 1986;44: 597–605. 5. Lemaitre J, Munting E, Mirtchi AA. Setting, hardening and resorption of calcium phosphate hydraulic cements. Rev Stomatol Chir Maxillofac 1992;93:163–5. 6. Hupp JR, McKenna SJ. Use of porous hydroxylapatite blocks for augmentation of atrophic mandibles. J Oral Maxillofac Surg 1988;46:538–45. 7. Kurashina K, Kurita H, Kotani A, Takeuchi H, Hirano M. In vivo study of a calcium phosphate cement consisting of alphatricalcium phosphate/dicalcium phosphate dibasic/tetracalcium phosphate monoxide. Biomaterials 1997;18:147–51. 8. Wittkampf AR. Fibrin glue as cement for HA-granules. J Craniomaxillofac Surg 1989;17:179–81. 9. Yokoyama A, Matsuno H, Yamamoto S, Kawasaki T, Kohgo T, Uo M, et al. Tissue response to a newly developed calcium phosphate cement containing succinic acid and carboxymethyl-chitin. J Biomed Mater Res 2003;64A:491–501. 10. Yamaguchi K, Hirano T, Yoshida G, Iwasaki K. Degradationresistant character of synthetic hydroxyapatite blocks filled in bone defects. Biomaterials 1995;16:983–5. 11. Klein CP, Driessen AA, de Groot K, van den Hooff A. Biodegradation behavior of various calcium phosphate materials in bone tissue. J Biomed Mater Res 1983;17: 769–84. 12. Komuro Y, Imazawa T. Use of calcium phosphate cement in crahiofacial surgery. Med Postgrad 2003;41:131–5. 13. Imazawa T, Komuro Y. Experimental study of biocompatibility, osteoconductivity and utility of self-hardening hydroxyapatite in the cranio-maxillo-facial area. J Jpn Soc Plast Reconstr Surg 2003;23:556–66. 14. Katthagen BD, Mittelmeier H. Experimental animal investigation of bone regeneration with collagen-apatite. Arch Orthop Trauma Surg 1984;103:291–302. 15. Klein CP, van der Lubbe HB, de Groot K. A plastic composite of alginate with calcium phosphate granulate as implant material: an in vivo study. Biomaterials 1987;8:308–10. 16. Doyle C, Tanner ET, Bonfield W. In vitro and in vivo evaluation of polyhydroxybutyrate and of polyhydroxybutyrate reinforced with hydroxyapatite. Biomaterials 1991;12: 841–7.
196 17. Yamaguchi I, Tokuchi K, Fukuzaki H, Koyama Y, Takakuda K, Monma H, et al. Preparation and microstructure analysis of chitosan/hydroxyapatite nanocomposites. J Biomed Mater Res 2001;55:20–7. 18. Maruyama M, Ito M. In vitro properties of a chitosan-bonded self-hardening paste with hydroxyapatite granules. J Biomed Mater Res 1996;32:527–32. 19. Gerentes P, Vachoud L, Doury J, Domard A. Study of a chitinbased gel as injectable material in periodontal surgery. Biomaterials 2002;23:1295–302. 20. Tokura S, Tamura H. O-carboxymethyl-chitin concentration in granulocytes during bone repair. Biomacromolecules 2001;2:417–21.
H. Uda et al. 21. Lemperle G, Ott H, Charrier U, Hecker J, Lemperle M. Related articles, links PMMA microspheres for intradermal implantation: Part I. Animal research. Ann Plast Surg 1991; 26:57–63. 22. Frayssinet P, Trouillet JL, Rouquet N, Azimus E, Autefage A. Osseointegration of macroporous calcium phosphate ceramics having a different chemical composition. Biomaterials 1993;14:423–9. 23. Klein CP, Patka P, den Hollander W. Macroporous calcium phosphate bioceramics in dog femora: a histological study of interface and biodegradation. Biomaterials 1989;10:59–62.
Experimental studies on hydroxyapatite powdercarboxymethyl chitin composite: injectable material for bone augmentation Hirokazu Udaa,*, Yasushi Sugawaraa, Masayoshi Nakasub a
Department of Plastic and Reconstructive Surgery, Jichi Medical School, Tochigi, Japan New Ceramics Department, Asahi Optical Co. Ltd, Tokyo, Japan
b
Received 11 January 2002; accepted 30 November 2004
KEYWORDS Hydroxyapatite; Carboxymethyl chitin; Bone augmentation; Physical gels
Summary We developed a hydroxyapatite (HA) powder-carboxymethyl chitin composite (HA-CMC composite) that can be injected with a 14G needle by adding distilled water. We prepared Materials I (HAZ57.0 wt%) and II (HAZ40.2 wt%) and examined their biocompatibility and osteoconductivity. With a 2-mm skin stab, the material was injected on the calvarial bone of rats. The periosteum was denuded blindly in half of the cases and preserved in the other half of the cases. Simultaneously, the material was injected subdermally into the abdominal skin to examine diachronic volume alteration of the material. Our results indicated that the new materials had biocompatibility as high as that achieved with previously developed HA materials. The difference in HA concentration did not influence the osteoconductivity, but the periosteum and the soft tissue on the cranium seemed to be an obstacle to bone ingrowth. On the other hand, the volume alteration was significantly smaller in Material I than in Material II. This composite may be especially useful in facial bone augmentation because it can be injected with only a small skin stab. When used for that purpose, the periosteum of the host bone should be denuded to facilitate bone ingrowth, and Material I will be preferable to Material II in terms of the maintenance of the initial volume. Q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved.
Bone substitute must be easy to shape, easy to handle, and able to maintain its initial volume. Hydroxyapatite (HA), as is now well known, has high biocompatibility and osteoconductivity, and it has
* Corresponding author. E-mail address: [email protected] (H. Uda).
been widely used as a bone substitute material in orthopaedic, periodontal, maxillofacial, and reconstructive surgery.1–4 However, some difficulties have been reported. HA blocks are usually difficult to shape to the desired form and troublesome to fit to the recipient side intraoperatively.4–6 HA granules can be applied to an uneven surface of a
S0007-1226/$ - see front matter Q 2005 The British Association of Plastic Surgeons. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.bjps.2004.11.022
Experimental studies on hydroxyapatite carboxymethyl chitin composite recipient bone, but are difficult to handle and may not retain their initial shape and volume because of secondary migration. 4,7,8 Calcium phosphate cement has been used in an attempt to overcome these disadvantages,7,9–11 but the effects of blood and tissue fluid at the recipient site prevents this material from transforming HA, leading to secondary migration and partial absorption.12 Thus, even calcium phosphate cement requires a dry field at the recipient site, and a sizeable skin incision is needed to achieve sufficient hemostasis.13 This skin incision limits the clinical use of HA, especially in facial bone augmentation. To address that problem, we have developed a hydroxyapatite powdercarboxymethyl chitin composite (HA-CMC composite). This system is dry and epispastic generally and can be gelled and injected with a 14G needle by adding distilled water (Fig. 1). In this system, the degradable carboxymethyl chitin (CM chitin) acts as an injectable material and binder to prevent the migration of HA grains from the recipient site. In this report, we described the preliminary development of HA-CMC composite, our observation of its biocompatibility histologically, and the results of our quantitative examination of its osteoconductivity by means of histomorphometry, as well as the rate of volume alteration after implantation by experiments on rats.
Materials and methods HA powder and CM chitin were obtained from Asahi Optical Co. (Tokyo, Japan). The diameter of the HA grain ranged from 100 to 300 mm, and the shape of the grain was inconstant (Fig. 2).
189
Figure 2 Electron micrograph of HA grains. The diameter of the HA grain ranged from 100 to 300 mm, and the shape of the grain was inconstant.
equal in the two materials as 2.0 percent weight by adding the proper amount of distilled water. By using two connected syringes, one filled with distilled water and another filled with HA-CMC composite, it was easy to achieve homogeneous distribution of the HA in the chitin gel (Fig. 3), and the homogeneous distribution could be maintained due to the viscosity of the chitin gel.
Implantation procedures We used female Wister rats, 6 to 8 weeks old. After mixing the HA-CMC composites and distilled water using a 5-ml syringe, we made a subdermal pocket in the head of each rat with a small 2-mm skin stab. The composite material was then injected with a 14G needle on the calvarial bone Fig. 4. At that time the periosteum was denuded blindly through the
Preparation of HA-CMC composite We prepared two kinds of HA-CMC composites, Materials I and II. Table 1 shows the amount of reagent in the preparation. The proportion of HA in Material I was higher than that in Material II (Material IZ57.0 wt%, Material IIZ40.2 wt%), and the concentrations of CM chitin were adjusted to be
Figure 1 The test material can be gelled and injected easily with a 14G needle by adding distilled water.
Figure 3 By using two connected syringes, one filled with distilled water and another filled with HA-CMC composites (a), it was easy to achieve homogeneous distribution of the HA in the chitin gel (b).
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Table 1
Material I Material II a
Preparation of HA-CMC composite in one vial HA (g)
CM chitin (g)a
Distilled water (ml)
Weight ratio of HA (wt%)
2.72 1.41
0.04 0.04
2 2
57.0 40.2
Adding 2 ml distilled water, the concentration of CM chitin solution was 2 wt% in each material.
skin stab with a small dissector in half of the cases, and it was preserved in the rest of the cases (Fig. 5). In addition, to examine diachronic volume alteration of the materials, we simultaneously injected 1 ml of the same material subdermally into the abdominal skin. Rats receiving these procedures were divided into the following groups (Table 2): Material I and periosteum was denuded (nZ16, group A), Material I and periosteum was preserved (nZ16, group B), Material II and periosteum was denuded (nZ16, group C), and Material II and periosteum was preserved (nZ16, group D).
Histological evaluation In all groups, the rats were sacrificed 2, 4, 8, or 16 weeks after implantation, respectively, (nZ4 at each time period). After macroscopic observation, implanted materials on the heads of rats were delivered en bloc with calvarial bones. The samples were placed immediately into 10% buffered formalin solution and examined histologically.
Histologic specimens were decalcified, embedded in paraffin, and cut into 4.5 mm thick sections just on the centre of the posterior frontal suture along the long axis of calvarial bones. The specimens were stained with hematoxylin and eosin and then examined with a light microscope. Next, they were diachronically examined for soft tissue reaction around the HA granules such as inflammation and its maturation. We also determined the bone ingrowth rates of all four groups by quantitative histomorphometric examination of osteoconductivity in all groups. With the histological sections at the sagittal plane just on the centre of the posterior frontal suture Fig. 6, of which the length was 3 mm and the height was 1.5 mm, we determined the areas of new bone ingrowth and calculated the bone ingrowth rates {(the area of new bone ingrowth/3!1.5 mm2)! 100(%)}. These were analysed using Adobe Photoshop 7.0 and NIH Image 1.63.
Evaluation of the rate of materials volume alteration In all groups, the materials injected subdermally into the abdominal skin were harvested simultaneously when the rats were sacrificed, and in each cases the volume of the materials was immediately measured with a volumeter to examine volume alteration of the materials. We examined the diachronic rates of volume alteration in Materials I and II.
Figure 4 A subdermal pocket was made in the head of each rat (patched area) with a small 2-mm skin stab. The composite material was then injected with a 14G needle on the calvarial bone.
Figure 5 During the injection of materials, the periosteum was preserved in half of the cases (a) and denuded blindly through the skin stab with a small dissector in the rest of the cases (b).
Experimental studies on hydroxyapatite carboxymethyl chitin composite Table 2
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Dividing groups
Group
A
B
C
D
Material Periosteum
I Torn off
I Preserved
II Torn off
II Preserved
The cases were divided into four groups. nZ16 in all.
Results Gross findings Even at 2 weeks after implantation, the head and nasal dorsum in which the material was injected were well augmented. When we dissected the skin and observed the material directly, the material was already covered entirely with a thin, pellucid capsule and the material had retained its initial shape. No secondary migration of HA grains was seen. These findings could be seen in all cases of groups A–D.
Histological findings In all groups, all interspaces between HA grains were filled with immature granulation tissue at 2 weeks after implantation, and each grain was surrounded by this tissue. We observed a slight infiltration of inflammatory cells. Simultaneously, some foreign body giant cells stuck to HA grains were defined (Fig. 7(a) and (b)). The granulation tissue, mainly composed by fibroblast, had been matured diachronically, and each HA grain was well fixed with mature collagen fibre and kept from migrating at 16 weeks. Foreign body giant cells still existed, but no englobement of HA grains was seen (Fig. 7(c) and (d)) (Figs. 7 and 8). In groups A and C, in which the periosteum was denuded blindly, we found immature granulation tissue in most of the contact area between the
material and the calvarial bone; we also saw a little new bone formation. However, at 4 weeks after implantation, there was additional new bone formation, and it had spread along the material surface facing the host bone (Fig. 8(a) and (b)). New bone seemed to increase with time. At 16 weeks after implantation, almost all contact surface of the material was bound with new bone, and partially deep ingrowth of the new bone was observed (Fig. 8(c) and (d)). Regarding the soft tissue response to the materials, it seemed that there were no apparent difference among groups A, B, C, and D. With respect to the hard tissue response to the materials, no bone ingrowth was seen in any cases of groups B and D, in which the periosteum was preserved, throughout all the test periods. Even 16 weeks after implantation, coarse connective tissue seemed to prevent new bone ingrowth to the material (Fig. 8(e)). In contrast, quantitative examination of osteoconductivity revealed that the bone ingrowth rate gained diachronically in the cases of groups A and C, in which the periosteum was denuded. Statistical analysis (using the Mann–Whitney U tests and assuming unequal variances, we compared the bone ingrowth rates at 2, 4, 8, and 16 weeks after implantation; statistical significance was defined as P!0.05) revealed there were no significant differences in the bone ingrowth rate between the cases of groups A and C at 2, 4, 8, and 16 weeks after implantation (Fig. 9). That is, the osteoconductivity did not depend on Materials I and II.
Volume alteration of the materials
Figure 6 The areas of new bone ingrowth were calculated with the histological sections, of which the length was 3 mm and the height was 1.5 mm just to the centre of the posterior frontal suture (PF: posterior frontal suture, Cor: coronal suture, Sag: sagittal suture, Lam: lamboid suture).
The volume of each material decreased uniformly at 2 weeks after implantation (Material I: meanZ 79%, Material II: meanZ63%). From 2 to 16 weeks after implantation, no significant volume alteration occurred in either material. As a whole, the volume of Material I was better retained than that of Material II. Statistical analysis (using the Mann– Whitney U test and assuming unequal variances, we compared the volume alterations of Materials I and II at 2, 4, 8, and 16 weeks after implantation; statistical significance was defined as P!0.05) between Materials I and II revealed that there
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Figure 7 Soft tissue response to the material in the case of group C (arrow: foreign body giant cell, *: HA grain). (a) and (b) At 2 weeks after implantation, the HA grains were surrounded by immature granulation tissues. A slight infiltration by inflammatory cells was observed, and some foreign body giant cells stuck to HA grain were defined (a: original magnification!40, b: original magnification!200). (c) and (d) At 16 weeks after implantation, the HA grains were well fixed with mature collagen fibre. Foreign body giant cells were still present, but englobement of HA grains was not seen (c: original magnification!40, d: original magnification!200).
were significant differences in volume alteration at 4, 8, and 16 weeks after implantation (Fig. 10).
Discussions HA has been widely used as a bone substitute material because of its high biocompatibility and osteoconductivity. However, for reconstructive and plastic surgeons who deal with facial bone, the originally developed HA materials are not a satisfying bone substitute, because in spite of recently developed calcium phosphate cement, a sizeable skin incision is needed for use of the material. This inevitable skin incision is a nuisance for facial bone augmentation. Our aim was to develop a facial bone augmentation material that can be injected without a sizeable skin incision. In our new material, HACMC composite, CM chitin acts to bind HA grains.
Previously, polymer-HA grain blends using binders such as collagen,14 alginate,15 polyhydroxybutyrate,16 fibrin glue,8 and chitosan17,18 had been reported, but these materials had various disadvantages. Some of them caused allergic reaction, some could not maintain homogeneity, some were excessively costly, some were difficult to preserve, some caused inflammation, and so on. As a result, these materials are not yet available clinically. In contrast, CM chitin, which is a derivative of chitin, may be suitable as a binder that can be used clinically. First, CM chitin is very economical, because chitin is one of the most abundant polymers in the biomass after cellulose and it is contained in crustaceans on a massive scale.19 Second, CM chitin is dry and epipastic in normal temperatures, so it is easy to preserve and easy to blend with HA grains. And last, CM chitin can be gelled easily by adding only distilled water, and it
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Figure 8 Hard tissue response to the material at the contact area between the material and the calvarial bone. Hematoxylin and eosin stain (*: HA grain, **: calvarial bone, arrow: new bone migration, arrowhead: periosteum). (a) and (b) At 4 weeks after implantation, in the case of group A, new bone spreading along the material surface facing the host bone was observed (a: original magnification!40, b: original magnification!100). (c) and (d) At 16 weeks after implantation, in the case of group A, almost all contact surface of the material was bound with new bone, and partially deep ingrowth of the new bone was observed (c: original magnification!40, d: original magnification!100). (e) At 16 weeks after implantation, in the case of group B, in which the periosteum was preserved, no new bone formation was seen even 16 weeks after implantation. Coarse connective tissue and the periosteum seemed to prevent new bone formation and its ingrowth to the material (original magnification!40).
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Figure 9 Quantitative examination of osteoconductivity revealed the bone ingrowth rate gained diachronically in the cases of groups A and C, in which the periosteum was denuded. Statistical analysis revealed there were no significant differences between the cases of groups A and C in the bone ingrowth rate at 2, 4, 8, and 16 weeks after implantation (using the Mann–Whitney U test and assuming unequal variances, we compared the bone ingrowth rates at 2, 4, 8, and 16 weeks after implantation; statistical significance was defined as P!0.05).
Figure 10 Statistical analysis (using the Mann–Whitney U test and assuming unequal variances, we compared the volume alterations of Materials I and II at 2, 4, 8, and 16 weeks after implantation) between Materials I and II revealed there were significant differences in volume alteration at 4, 8, and 16 weeks after implantation (*statistical significance was defined as P!0.05).
H. Uda et al. can maintain that property for a long time, unlike chitosan, which needs chemical modification with anhydrides or aldehydes to gelify.19 Moreover, the deacetylisation rate of our CM chitin is zero. Tokura and Tamura20 reported that the amino group in chitosan causes a bioactive reaction in the animal body, and in the same way the cytotoxicity of CM chitin is in proportion to the deacetylation rate of CM chitin. These differences indicate that our CM chitin material is superior to chitosan gel in regard to cytotoxicity. In this study, macroscopic observation showed satisfactory results of bone augmentation with the composite we developed. At 2 weeks after implantation, the material was entirely covered by a thin capsule and well fixed. Secondary migration of the material was not observed in any cases. At the same time, histological findings at two weeks after implantation revealed that each HA grain was enclosed and fixed by immature granulation tissue that seemed to prevent HA grains from migrating. These findings proved that CM chitin worked not only as an injectable binder of HA grains but also as a temporary scaffold. The granulation tissue matured gradually, and at 16 weeks after implantation it turned to mature collagen fibre, which in turn further fixed each HA grain. This finding proved that our system has the same high biocompatibility as that of previously developed HA substitutes. We found that the concentration of HA did not influence osteoconductivity in our material. In opposition to that, the results in groups A and C, in which the periosteum of the host bone was denuded blindly, were apparently superior to the results in groups B and D, in which the periosteum was preserved. We hypothesised that coarse connective tissue between the material and the host bone prevented new bone formation and bone ingrowth in the cases of which the periosteum was preserved. It should be noted that injury to the periosteum stimulates the cellular events that lead to passive bone ingrowth at the bone-implant interface. For bone augmentation, material should bond tightly to the recipient bone surface. We, therefore, believe the periosteum should be denuded when using this material clinically. As to volume decrease, we suspect that the early volume decrease of the material at 2 weeks after implantation (Material I: meanZ79%; Material II: meanZ63%) is due to absorption of water and CMchitin. After that, from 2 to 16 weeks, the volumes of both materials were well preserved. An ability to preserve initial volume is an invaluable property for bone augmentation material. In that respect, Material I was superior to Material II. The preservative capacity of the initial volume seemed to
Experimental studies on hydroxyapatite carboxymethyl chitin composite be in proportion to the HA rate of the composite. However, it was difficult to gain an HA rate higher than that of Material I, because injection with a 14G needle became impossible with higher rates. That problem may be solved by shrinking the HA grains or by changing the inconstant shape of the HA grains to a rounded shape. Additional resorption of material may occur in the long term, following the 16-week limit of our testing. In our results, histological findings at 16 weeks after implantation revealed that foreign body giant cells still existed around HA grains. Particles up to 15 mm can avoid phagocytosis;21 our HA grains were 100–300 mm to prevent englobement by phagocytes, but many minute fragments of HA grains smaller than 100 mm were actually observed. We supposed these fragments were mainly the result of physical demolition that occurred during when mixing of the composites and water with a syringe. These minute fragments might be digested in the long term, though englobement of HA grains was not seen in our histological sections at 16 weeks after implantation. Moreover, it has also been reported that phagolytic cells resorb calcium phosphate ceramics by two processes of dissolution and digestion.7,22,23 A much longer follow-up study may be needed to evaluate volume alteration of our material. Our material should be useful clinically, especially in cranio-maxillofacial surgery, as in, for example, revision of deformity after facial bone fracture and treatment for minor deformity of facial anomalies. Moreover, our system will be attractive in cases of cosmetic surgery, such as augmentation rhinoplasty, genioplasty, and malar bone augmentation, because only a small skin stab will be needed to administer the material. Based on the results of our study, we caution users that in those cases the periosteum of the host bone should be denuded by a small dissector to promote the osteoconductivity of the material, and the rate of HA grains should be higher (that is, Material I is superior to Material II). A few disadvantages became clear from our experience. First, we had difficulty forming this material into a detailed shape, because the shape depended on the shape of the subcutaneous pocket to some degree. Second, this composite is not selfhardening bone cement, so some period (about 5 to 7 days experimentally in this study) was necessary to fix the composite by fibrous capsule. And last, a certain decrease of initial volume, especially within the initial 2 weeks, occurred as mentioned above. There is room for improvement in our material. For instance, we would like to increase the rate of HA that can maintain its initial volume while also
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maintaining the material’s injectable property, and we would like to be able to prevent the generation of minute HA fragments due to physical demolition to protect against phagocytosis. However, we think this HA-CMC composite has good potential for expanded clinical use, especially for facial bone augmentation, because of its high biocompatibility, good osteoconductivity, easy handling, low cost, and easy preservation.
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