Tissue responses of calcium phosphate cement: a study in dogs

The in vivo properties of a new kind of calcium phosphate cement were investigated in this study. Calcium phosphate cement was implanted as paste into femoral bone and dorsal muscle of dogs for 3 and 6 months, and as prehardened form into thigh muscles of dogs for 1, 2 and 6 months. Histology was performed on thin un-decalcified sections. No foreign body reaction, no inflammation and no necrosis were found both in bony site and in muscles. There was no connective tissue layer between the cement and bone when cement paste was implanted in the bone. A creeping substitution of cement by bone, in which osteoclast-like cells resorbed the cement as if the cement is a part of bone and new bone was formed directly on the resorption line of calcium phosphate cement, was found. Bone formation was found histomorphologically in pores and deep rugged surface of cement samples (both paste and prehardened form) implanted in muscles of dogs. The induced bone was also identified with backscattered scanning electron microscopy (BSE) and by energy-dispersive X-ray micro-analysis (EDX). The results suggest that the calcium phosphate cement used in this study is biocompatible, resorbable in a manner of creeping substitution, osteoconductive and osteoinductive. It seems that an ideal bone substitute can be developed by using this type of calcium phosphate cement.     

Design of injectable organic-inorganic hybrid for bone tissue repair

Injectable bone substitutes are rapidly gained success in tissue engineering applications for their less invasive surgical aspect. Here, the design and the characterization of a novel degradable paste of PCL reinforced with nanocrystals of hydroxyapatite have been presented aiming to mimic natural tissue. Nanohydroxyapatite has been successfully synthesized via sol-gel technique. Dynamic and steady state viscoelastic properties of the solutions and paste were investigated to control the kinetic of phase transition. Correspondingly, the morphology and composition were characterized via TEM, EDAX, and thermal analysis. Injection test underlines the completely ability of the paste of being injected without altering its features. Preliminary biological study showed that the composite paste is not cytotoxic. The synergistic rheological and biological properties, combined with the positive effect of chemical synthesis method indicate that the composite paste is very suitable as local bone substitute in low-load areas.     

Calvarial bone response to a tricalcium phosphate-genipin crosslinked gelatin composite

A biodegradable composite which was composed of genipin cross-linked gelatin mixing with tricalcium phosphate ceramic particles (GGT) was developed as a bone substitute. This study was evaluated by the biological response of rabbit calvarial bone to assess the potential of the GGT composite as a biodegradable and osteoconductive bone substitute. Eighteen New Zealand white rabbits were used for cranial implantation. Bone defects (15 x 15 mm) of nine rabbits were filled with the GGT composites, while the others were filled with the de-proteinized bovine bones as controls. Three rabbits were examined for each group in every time period at 4, 8 and 12 weeks post-surgery. The assessment included serial post-operative gross examinations, radiographic analyses and histological evaluations. This study demonstrated that this composite is: (1) malleable, with easily molded to the calvarial bone defect without fracture; (2) biocompatible, with no evidence of adverse tissue reaction; (3) osteoconductive, with progressive growth of new bone into the calvarial bone defect; (4) biodegradable, with progressive replacement of the composite by new bone. Additionally, results of both radiographic analyses and histological evaluations revealed obviously greater new bone ingrowth in the GGT composite compared with the de-proteinized bovine bone at the same implantation time. Therefore, the GGT composite could serve as a useful bone substitute for repairing bone defects.     

In vivo evaluation of a porous hydroxyapatite/poly-DL-lactide composite for bone tissue engineering

As reported previously, a porous composite of uncalcined hydroxyapatite (u-HA) and poly-DL-lactide (PDLLA) showed excellent osteoconductivity and biodegradability as a bone substitute in rabbit model. In this study, to investigate the usefulness of this composite as a scaffold loaded with cells, we estimated whether this material showed osteogenesis on implantation to extraosseous site. On loading with syngeneic bone marrow cells and implantation into rat dorsal subcutaneous tissue, osteogenesis with enchondral ossification was seen both on and in the material at 3 weeks after implantation. The osteogenesis in the u-HA/PDLLA had progressed, and newly formed bone tissue was found in the material by 6 weeks. To investigate the osteoinductive properties of the material, we implanted this porous composite material into extraosseous canine dorsal muscle. At 8 weeks, osteogenesis was seen in the pores of the material. Newly formed bone could be observed adjacent to the material. In addition, cuboidal osteoblasts adjacent to the newly formed bone were evident. Neither cartilage nor chondrocytes were found. These results might indicate that the material induced osteogenesis by intramembranous ossification. Conversely, similar porous PDLLA did not induce osteogenesis during the observation period. Therefore, porous HA/PDLLA, which has osteoconductive and osteoinductive properties, might be a useful material for use as a bone substitute and cellular scaffold.     

Brushite-collagen composites for bone regeneration

Brushite-based biomaterials are of special interest in bone regeneration due to their biocompatibility and biodegradability; on the other hand, collagen is a well-known osteoconductive biomaterial. In the present study a new brushite-collagen composite biomaterial is reported. This new biomaterial was prepared by combining citric acid/collagen type I solutions with a brushite cement powder. The obtained biomaterial was a cement paste, with improved handling properties. The effect of collagen on the setting reaction of brushite cement was studied, and was found to speed up the cement setting reaction. The cement paste set into a hard ceramic material within 18.5+/-2.1min and had compressive strength similar to that of spongeous bone (48.9+/-5.9MPa in dry conditions and 12.7+/-1.5MPa in humid conditions). The combination of collagen with citric acid revealed an interesting synergistic effect on the compressive strength of the composite material. Moreover, this new biomaterial had excellent cohesion properties (ninefold better than brushite cement), and high cellular adhesion capacity (threefold higher than brushite cement). The composite biomaterial described in this study combines good handling properties, compressive strength, cohesion and cell adhesion capacity, along with the osteoconductive and biodegradable properties inherent in brushite and in collagen-based biomaterials.     

Histological evaluation of the bone response to calcium phosphate cement implanted in cortical bone

The aim of this study was to investigate the physicochemical and biological properties of a newly developed calcium phosphate cement (CaP cement) implanted in cortical bone. CaP cement was injected as a paste into tibia cortical bone defects in goats. Polymethylmethacrylate (PMMA) bone cement was used as a control. The animals were killed after 3 days, 2, 8, 16 and 24 weeks. X-ray diffraction and Fourier transform infrared spectroscopy performed at retrieved samples showed that the CaP cement had set as a carbonate apatite and remained stable over time. Light microscopic evaluation showed that after 2 weeks the cement was in tight contact with the bone without any inflammatory reaction or fibrous encapsulation. At later time points, the CaP cement implants were totally covered by a thin layer of bone and osteoclasts, present at the interface, which were clearly resorbing the cement. At locations where CaP cement was resorbed, new bone was deposited. Transmission electron microscopy revealed that indeed a seamless contact existed between CaP cement and bone, as characterized by the occurrence of an electron dense line of 50-60 nm thick that covered the CaP cement. Osteoblasts, in contact with the cement, were depositing new bone. Although the bulk of the material was still in situ after 24 weeks, the progressive osteoclast resorption of the cement followed by new bone formation suggests that all of the material may be replaced eventually. In contrast to the CaP cement, the PMMA reference cement was always surrounded by a thin fibrous capsule. The results indicate that the investigated CaP cement is biocompatible, osteoconductive as well as osteotransductive and is a candidate material for use as a bone substitute.     

In vivo evaluation of hydroxyapatite foams

Hydroxyapatite (HA) is widely applied as bone graft material due to its osteoconductive potential and well-established biocompatibility. In this work, macroporous hydroxyapatite structures made through foaming of aqueous suspensions and gelcasting were tested for in vivo osteointegration. These foams are composed of a three-dimensional array of spherical pores with diameters of approximately 100-500 microm, interconnected by windows of smaller size in the range of 30-120 microm. The HA foams were implanted in the tibia of albino New Zealand rabbits and removed after a period of 8 weeks. Histological analysis revealed that the pores in the foams were partially or completely filled progressively with mature new bone tissue and osteoid after the implanted period. No immune or inflammatory reactions were detected. The high osteoconductive potential of the HA foams provides a potential structure for use as bone substitute in orthopedic, oral, and cranio-maxillofacial reconstructive surgery, and as dento-alveolar implants.     

Histological examination of beta-tricalcium phosphate graft in human femur

Prominent osteoconductive activity and the biodegradable nature of beta tricalcium phosphate (beta-TCP) for bone grafts in animal experiments has been reported. A new type of beta-TCP has been manufactured at extraordinarily high purity and has been available as potent bone grafting substitute for clinical use. The histological features of grafted beta-TCP in human bone have been analyzed. A 33-year-old female with a bone tumor of the proximal femur underwent curettage and beta-TCP graft under the diagnosis of probable benign fibrous dysplasia. Four weeks later, the proximal femur, including the grafted beta-TCP was resected because of the final diagnosis of the curettaged materials was osteosarcoma. The resected specimen revealed abundant direct new bone apposition on beta-TCP. There was no cartilaginous tissue or enchondral ossification. Bone formation was more prominent in the periphery of the grafted area than in the center. There was a considerable number of osteoclast-like giant cells surrounding the beta-TCP. This case illustrated that highly purified beta-TCP had prominent osteoconductive activity and biodegradable nature in human bone.     

Beta-TCP bone graft substitutes in a bilateral rabbit tibial defect model

The use of artificial bone graft substitutes has increased as the surgical applications widen and the availability of allograft bone decreases. The ideal graft substitute should reabsorb with time to allow and encourage new bone formation whilst maintaining its properties as an osteoconductive scaffold until it is no longer required. A potential disadvantage of some synthetic substitutes is their long dissolution time. Beta-tricalcium phosphates (beta-TCPs) have some advantages when compared to hydroxyapatite (HA), when used as a filler, in that it is more rapidly reabsorbed. Three commercially available and clinically used beta-TCP bone graft substitutes with the same chemistry (Vitoss, Osferion, Chronos) but with varying macro and microscopic characteristics were investigated using a bilateral tibial metaphyseal defect model in New Zealand white rabbits. When placed into tibial defects all three materials performed similarly in terms of mechanical properties of the healing defects. A decrease in properties was found at 12 weeks where implant resorption was nearly achieved while remodelling of the anteromedial cortex had yet to be completed. All materials were osteoconductive and supported new bone formation while implant resorption with time differed between materials. Vitoss resorbed faster than the other materials and is likely to differences in particle geometry, pore structure and interconnectivity.     

Trabecular bone response to injectable calcium phosphate (Ca-P) cement

The aim of this study was to investigate the physicochemical, biological, and handling properties of a new developed calcium phosphate (Ca-P) cement when implanted in trabecular bone. Ca-P cement consisting of a powder and a liquid phase was implanted as a paste into femoral trabecular bone of goats for 3 days and 2, 8, 16, and 24 weeks. The cement was tested using three clinically relevant liquid-to-powder ratios. Polymethylmethacrylate bone cement, routinely used in orthopedics, was used as a control. The Ca-P cement was easy to handle and was fast setting with good cohesion when in contact with body fluids. X-ray diffraction at the different implantation periods showed that the cement had set as an apatite and remained stable over time. Histological evaluation after 2 weeks, performed on 10 microm un-decalcified sections, showed abundant bone apposition on the cement surface without any inflammatory reaction or fibrous encapsulation. At later time points, the Ca-P cement implants were totally covered by a thin layer of bone. Osteoclast-like cells, as present at the interface, had resorbed parts of the cement mass. At locations where Ca-P cement was resorbed, new bone was formed without loss of integrity between the bone bed and the cement. This demonstrated the osteotransductive property of the cement, i.e., resorption of the material by osteoclast-like cells, directly followed by the formation of new bone. Histological and histomorphometrical evaluation did not show any significant differences between the Ca-P cement implanted at the three different liquid/powder ratios. The results indicate that the investigated Ca-P cement is biocompatible, osteoconductive, as well as osteotransductive and is a candidate material for use as a bone substitute.     

Injectable nanocrystalline hydroxyapatite paste for bone substitution: in vivo analysis of biocompatibility and vascularization

The nanocrystalline hydroxyapatite paste Ostim represents a fully degradable synthetic bone substitute for the filling of bone defects. Herein, we investigated in vivo the inflammatory and angiogenic host tissue response to this biomaterial after implantation. For this purpose, Ostim was implanted into the dorsal skinfold chambers of Syrian golden hamsters. The hydroxyapatite ceramic Cerabone and isogeneic transplanted cancellous bone served as controls. Angiogenesis, microhemodynamics, microvascular permeability, and leukocyte-endothelial cell interaction of the host tissue were analyzed over 2 weeks using intravital fluorescence microscopy. Ostim exhibited good biocompatibility comparable to that of Cerabone and cancellous bone, as indicated by a lack of venular leukocyte activation after implantation. Cancellous bone induced a more pronounced angiogenic response and an increased microvessel density when compared with the synthetic bone substitutes. In contrast to Cerabone, however, Ostim showed a guided neovascularization directed toward areas of degradation. Histology confirmed the ingrowth of proliferating vascularized tissue into the hydroxyapatite paste at sites of degradation, while the hydroxyapatite ceramic was not pierced by new microvessels. Thus, Ostim represents an injectable synthetic bone substitute, which may optimize the conditions for the formation of new bone at sites of bone defects by supporting a guided vascularization during biodegradation.     

Tissue response to porous high density polyethylene as a three-dimensional scaffold for bone tissue engineering: An experimental study

High density polyethylene (HDPE) is a synthetic biomaterial used as a three-dimensional scaffold for bone defect reconstruction. Reports differ with regard to its biological response, particularly its osteoconductive capacity. The aim of the present work was to histologically and histomorphometrically evaluate tissue response to porous HDPE. An in vivo study was conducted in rat tibia to evaluate osteogenic capacity, angiogenesis, inflammatory response, and the presence of multinucleated giant cells 14 and 60?days post-biomaterial implantation. Histological examination 14?days post-implantation showed fibrovascular tissue inside pores and on the surface of porous HDPE, acute inflammatory response, scant multinucleated giant cells (MNGCs), and lamellar bone in contact with the biomaterial. An increase in the proportion of lamellar bone tissue, no inflammatory response, and a decrease in the number of MNGCs were observed at 60?days. The histomorphometric study showed a significant time-dependent increase both in the area of bone tissue formed in contact with the porous HDPE (14d: 24.450?±?11.623 µm² vs. 60d: 77.104?±?26.217 µm², p?<?0.05) and in the percentage of bone tissue in contact with the porous HDPE (osseointegration). A significant decrease in the number of MNGCs was also observed at 60?days post-implantation. Porous HDPE showed adequate osteoconductive properties, and only caused an initial inflammatory response. Although this biomaterial has traditionally been used juxtaosseoulsy, its adequate osteoconductive properties broaden the scope of its application to include intraosseous placement.     

无细胞骨胶原基质的理化性能和组织相容性研究

制备了一种无细胞骨胶原基质(Acellular bone collagen matrix,ABCM)和ABCM-PDLLA复合材料。对材料的各种理化性能进行测试,并通过酶联免疫吸附(Enzyme linked immunosorbent assay,ELISA)和兔桡骨骨干缺损修复实验评价材料的组织相容性。结果表明材料中主要的骨矿成分和有机成分分别是碳酸盐羟基磷灰石和胶原,脂肪和细胞成分被完全脱除;ABCM材料具有较好的力学强度和微孔结构,加入PDLLA有助于提高材料的力学性能;ELISA检测显示材料引起的免疫反应持续时间较短,加入PDLLA可降低免疫反应。体内移植实验表明材料主要以传导成骨方式实现骨的修复和替代。总之,两种材料均具有较好的组织相容性,ABCM-PDLLA具有更好的力学性能和较低的免疫原性,更适合用作骨移植材料或骨组织工程支架材料。     

In vitro and in vivo evaluation of a novel nanosize hydroxyapatite particles/poly(ester-urethane) composite scaffold for bone tissue engineering

Scaffolds for bone tissue engineering should provide an osteoconductive surface to promote the ingrowth of new bone after implantation into bone defects. This may be achieved by hydroxyapatite loading of distinct scaffold biomaterials. Herein, we analyzed the in vitro and in vivo properties of a novel nanosize hydroxyapatite particles/poly(ester-urethane) (nHA/PU) composite scaffold which was prepared by a salt leaching–phase inverse process. Microtomography, scanning electron microscopy and X-ray spectroscopy analyses demonstrated the capability of the material processing to create a three-dimensional porous PU scaffold with nHA on the surface. Compared to nHA-free PU scaffolds (control), this modified scaffold type induced a significant increase in in vitro adsorption of model proteins. In vivo analysis of the inflammatory and angiogenic host tissue response to implanted nHA/PU scaffolds in the dorsal skinfold chamber model indicated that the incorporation of nHA particles into the scaffold material did not affect biocompatibility and vascularization when compared to control scaffolds. Thus, nHA/PU composite scaffolds represent a promising new type of scaffold for bone tissue engineering, combining the flexible material properties of PU with the advantage of an osteoconductive surface.     

骨组织工程中支架材料的研究进展

骨组织工程的支架材料首先应具有支持结构,其次具有组织相容和引导成骨的作用。目前骨组织工程的支架材料种类很多、来源不同、性能各异。本文综述了骨组织工程中支架材料的最新研究进展和前景。     

Simultaneous in vivo comparison of bone substitutes in a guided bone regeneration model

A direct, simultaneous comparison of bone substitutes is hampered by the limited number of samples that can be tested simultaneously. The goal of this study was to establish a preclinical model for guided bone regeneration that offers testing of different bone substitutes in a one-wall defect situation. We show here that up to eight titanium hemispheres can be placed on the calvaria of minipigs. To establish our model, titanium hemispheres were filled with and without Bio-Oss, a deproteinized bovine bone mineral, Ostim, an aqueous paste of synthetic nanoparticular hydroxyapatite, and Osteoinductal, an oily calcium hydroxide suspension, before being positioned on the calvaria. After 6 and 12 weeks, titanium hemispheres were subjected to histological and histomorphometric analysis. We show here that bone filled approximately one-tenth of the area below the hemispheres which were left empty, indicating a critical size model for guided bone regeneration. In accordance with the documented osteoconductive properties of Bio-Oss and Ostim, titanium hemispheres were almost completely filled with bone. Moreover, the expected degradation profile of Bio-Oss and Ostim could be confirmed by histologic and histomorphometric analysis. Under the same conditions, Osteoinductal failed to exert osteoconductive properties, rather a progressive resorption of the host bone was observed. These results demonstrate that the preclinical model presented here is suitable to simultaneously compare bone substitutes with different material properties. Our model based on the titanium hemispheres allows evaluation of graft consolidation under standardized conditions thereby avoiding intra-individual variations.     

Effects of additives on the rheological properties and injectability of a calcium phosphate bone substitute material

An injectable calcium phosphate bone substitute material has been prepared by mixing amorphous calcium phosphate (ACP) and dicalcium phosphate dihydrate (DCPD) for use in noninvasive surgery, and the influence of additives, such as disodium hydrogen phosphate, polyethylene glycol (PEG), glycerin, and citric acid, on the rheological properties and injectability of the ACP + DCPD cement system have been studied in this work. Novel approach of thixotropy measurement has been used to characterize the stability of the pastes. The results show that the injectability and the setting time can be augmented by the addition of disodium phosphate solution to the paste but reduced by the addition of PEG 200, glycerin, or citric acid to the paste. This study suggests that the injectability and the setting time of the ACP + DCPD bone substitute material can be balanced, and the injectable calcium phosphate bone substitute material with satisfied fluidity and injectability for clinical operation can be prepared by optimizing the additives and their concentrations, according to different clinical requirements.     

Evaluation of bone healing with eggshell-derived bone graft substitutes in rat calvaria: a pilot study

The purpose of this study was to investigate the potential effectiveness of a surface-modified natural calcium carbonate, hen eggshell (ES) as a bone graft substitute. The surface characteristics, cell viability on, and osteoconductivity of, particulated ES with and without hydrothermal treatment in phosphate solutions were evaluated. Hydrothermal treatment partially converted ES to calcium-deficient hydroxyapatite (HA) with surface microstructure. MTT assay indicated higher osteoblast viability on surface-modified ES compared with a commercially available bone substitute, anorganic bovine bone (Bio-Oss, BO) (p < 0.001). Histological and histomorphometric analysis showed significantly greater new bone formation and mineralized bone-to-graft contact of surface-modified ES, especially with hydrothermally treated ES, compared with BO in 5-mm diameter calvarial defects in rats at 4 and 8 weeks of healing (p < 0.01). Complete bony bridging was more frequently found with hydrothermally treated ES. The results of this pilot study indicate the potential efficacy of surface-modified particulated hen eggshell as an osteoconductive bone substitute in a rat calvarial defect model.     

Osteointegration of orthopaedic devices

The different properties of bone must be considered in order to understand the relation between orthopaedic devices and bone. The epi-/metaphyseal areas are defined by their rigidity, their high vascularity and their quick remodelling process. In contrast, the diaphyses of bone are rather elastic and built of dense, scarcely vascularised bone presenting slow remodelling. Implants can integrate by pure mechanical contact without real affinity to bone or, alternatively, they can favour ongrowth of bone, provided that they are osteoconductive. Amongst different bone substitutes, only some of them are absorbable. Only derivates of bone may present the property of osteoinduction, which is the power to create new bone in any region of the body. Orthopaedic devices are characterised by their shape, their stiffness or elasticity and by the characteristic properties of material. They may be osteoconductive such as titanium alloys and some ceramics, allowing integration in bone. Alternatively, other materials such as steel, CoCr alloys and PMMA cements remain separated from bone by a tiny layer of collagen. The surface structure influences the quality of integration. The integration of implants depends on the mutual interaction of the material with the tissue on the implantation site. All implants undergo fatiguing which can lead to fracture of the implant. All implant–bone contacts are threatened by granulation tissue mainly formed because of wear products, infection and other reasons.     

The effect of BMP-2 on the osteoconductive properties of β-tricalcium phosphate in rat calvaria defects

Bone formation in critical-sized calvaria defects is strongly dependent on the osteoconductive properties of grafts. It remains a matter of controversy whether biomaterials can replace autografts and whether the supplementation of biomaterials with Bone Morphogenetic Proteins (BMPs) is necessary to enhance bone formation. We examined rat calvaria critical-sized defects (5-mm-diameter) treated with β-tricalcium phosphate (TCP; Cerasorb? M), polylactic and polyglycolic acid gel (PLA/PGA; Fisiograft?) and calcium phosphate cement (CPC; Norian? CRS?), either alone or in the presence of 5 μg of BMP-2 after 45 days. Autografts and untreated defects served as controls. Bone formation was evaluated based on μCT analysis, histomorphometric analysis and fluorescence analysis. We report that TCP supported bone formation more efficiently than did autografts. Bone formation in the presence of TCP alone reached a maximal level, as BMP-2 supplementation failed to enhance bone formation. By contrast, no significant difference in bone formation was observed when PLA/PGA and CPC were compared to autografts. Moreover, the presence of BMP-2 did not substantially change the osteoconductive properties of PLA/PGA or CPC. We conclude that the osteoconductive properties of TCP are superior to those of autografts and that TCP does not require BMP-2 supplementation. Our findings also show that the decreased osteoconductive properties of PLA/PGA and CPC cannot be overcome by BMP-2 supplementation in rat calvaria defects.