Hierarchically biomimetic bone scaffold materials: nano-HA/collagen/PLA composite

A bone scaffold material (nano-HA/ collagen/PLA composite) was developed by biomimetic synthesis. It shows some features of natural bone both in main composition and hierarchical microstructure. Nano-hydroxyapatite and collagen assembled into mineralized fibril. The three-dimensional porous scaffold materials mimic the microstructure of cancellous bone. Cell culture and animal model tests showed that the composite material is bioactive. The osteoblasts were separated from the neonatal rat calvaria. Osteoblasts adhered, spread, and proliferated throughout the pores of the scaffold material within a week. A 15-mm segmental defect model in the radius of the rabbit was used to evaluate the bone-remodeling ability of the composite. Combined with 0.5 mg rhBMP-2, the material block was implanted into the defect. The segmental defect was integrated 12 weeks after surgery, and the implanted composite was partially substituted by new bone tissue. This scaffold composite has promise for the clinical repair of large bony defects according to the principles of bone tissue engineering.     

仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原的制备及其细胞相容性

背景：目前骨组织工程常用的支架材料主要有无机材料、有机高分子材料及天然衍生材料等,上述材料各有优缺点,为了充分发挥各类材料的优势,弥补其不足,目前多采用联合材料制备复合支架。 目的：制备新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原,并观察其对骨髓间充质干细胞增殖、黏附及分化的影响。 方法：制备壳聚糖/纳米羟基磷灰石/胶原复合支架材料,扫描电镜观察支架材料表面微观形貌;采用真空吸附法将骨形态发生蛋白7多肽与支架材料复合,高效液相色谱仪检测骨形态发生蛋白7多肽在体外的释放规律;将骨髓间充质干细胞接种到复合骨形态发生蛋白7多肽的仿生支架材料上,以未复合多肽的支架材料作为对照,检测支架材料表面细胞增殖、黏附率、生长形态及碱性磷酸酶活性。 结果与结论：壳聚糖/纳米羟基磷灰石/胶原支架材料呈多孔状,孔径10~100 µm;骨形态发生蛋白7多肽可以从支架材料中缓慢释出;在复合多肽的仿生支架材料表面,骨髓间充质干细胞的黏附及向成骨细胞方向分化能力均明显强于对照组(P < 0.05),而增殖能力与对照组差异无显著性意义(P > 0.05)。说明新型仿生支架材料骨形态发生蛋白7多肽/壳聚糖/纳米羟基磷灰石/胶原是一种理想的骨组织工程支架材料,具有良好的细胞相容性。     

胶原-壳聚糖制备仿生多层结构软骨支架

目的制备结构与天然软骨结构相似的仿生多层软骨支架。方法采用先后于-20℃和液氮中冷冻的预冻方式,冷冻干燥法制备双层支架。采用-20℃冷冻后,部分熔融再液氮重冻的预冻方式,冷冻干燥制备了厚度约2mm的仿生多层软骨支架。采用XRD和红外光谱观察胶原和壳聚糖的复合情况。采用SEM观察支架的形貌。对比了纯壳聚糖支架、纯胶原支架、胶原壳聚糖复合材料单层支架和仿生多层支架在干燥和湿润两种状态下的力学性能。结果胶原和壳聚糖的复合存在化学反应,复合材料形成更好的孔结构,仿生多层支架从上至下分别具有致密层结构,圆形孔结构和垂直孔结构。支架材料在干燥和湿润状态下的力学性能有很大差别,仿生多层支架在湿润状态下各层具有不同的力学性能。结论仿生多层软骨支架的结构接近于天然关节软骨多层结构,且在湿润状态下各层的力学性能有差异,有望更好地维持软骨细胞表型和提高软骨损伤修复效果。     

Collagen-hydroxyapatite composite prepared by biomimetic process

A novel bone graft substitute comprising a porous, collagenous scaffold was biomimetically coated with hydroxyapatite using a simulated body fluid solution chemistry method. The scaffold had a porosity of approximately 85%, with pore sizes between 30 microm and 100 microm. Glutaraldehyde vapor was used to stabilize the collagenous scaffold, giving a significantly increased thermal stability over an unstabilized scaffold, as shown by differential scanning calorimetry. A thin layer (<10 microm) of crystalline hydroxyapatite was deposited onto the stabilized collagenous scaffold by soaking the collagenous construct in simulated body fluid in the presence of calcium silicate glass. The presence of crystalline hydroxyapatite was confirmed by X-ray diffraction, energy-dispersive X-ray spectroscopy, and scanning electron microscopy. In vitro cytotoxicity testing of the composite construct using L-929 fibroblasts (ISO 10993-5) and rabbit periosteal cells revealed a cytocompatible material that supported cellular attachment and proliferation.     

Preparation of bone-like apatite-collagen nanocomposites by a biomimetic process with phosphorylated collagen

By imitating in vivo bone mineralization, bone-like apatite-collagen nanocomposites were prepared by chemical phosphorylation of collagen and subsequent biomimetic growth of bone-like nanoapatite on collagen nanofibers. Two steps were employed in the composites preparation. First, the collagen was phosphorylated by chemical treatment, which provides the nucleation sites for bone-like apatite mineralization. The subsequent growth of bone-like nanoapatite on the phosphorylated collagen nanofibers was performed in simulated body fluid (SBF). The characterization of the composites showed that the composites were composed of nanoapatite mineralized collagen nanofibers that exhibit similarity to natural bone in composition and crystal morphology.     

Preparation of a bonelike apatite-polymer fiber composite using a simple biomimetic process

A bonelike apatite-polymer fiber composite may be useful as an implant material to replace bone, the enthesis of a tendon, and the joint part of a ligament. We treated an ethylene-vinyl alcohol copolymer (EVOH) plate and knitted EVOH fibers with an oxygen plasma to produce oxygen-containing functional groups on their surfaces. The plasma-treated samples were alternately dipped in alcoholic calcium and phosphate ion solutions three times to deposit apatite precursors onto their surfaces. The surface-modified samples formed a dense and uniform bonelike surface apatite layer after immersion for 24 h in a simulated body fluid with ion concentrations approximately equal to those of human blood plasma. The adhesive strength between the apatite layer and the sample's surface increased with increasing power density of the oxygen plasma. The apatite-EVOH fiber composite obtained by our process has similarities to natural bone in that apatite crystals are deposited on organic polymer fibers. The resulting composite would possess osteoconductivity due to the apatite phase. With proper polymer selection and optimized synthesis techniques, a composite could be made that would have bonelike mechanical properties. Hence, the present surface modification and coating process would be a promising route to obtain new implant materials with bonelike mechanical properties and osteoconductivity.     

Design of graded biomimetic osteochondral composite scaffolds

With the ultimate goal to generate suitable materials for the repair of osteochondral defects, in this work we aimed at developing composite osteochondral scaffolds organized in different integrated layers, with features which are biomimetic for articular cartilage and subchondral bone and can differentially support formation of such tissues. A biologically inspired mineralization process was first developed to nucleate Mg-doped hydroxyapatite crystals on type I collagen fibers during their self-assembling. The resulting mineral phase was non-stoichiometric and amorphous, resembling chemico-physical features of newly deposited, natural bone matrix. A graded material was then generated, consisting of (i) a lower layer of the developed biomineralized collagen, corresponding to the subchondral bone, (ii) an upper layer of hyaluronic acid-charged collagen, mimicking the cartilaginous region, and (iii) an intermediate layer of the same nature as the biomineralized collagen, but with a lower extent of mineral, resembling the tidemark. The layers were stacked and freeze-dried to obtain an integrated monolithic composite. Culture of the material for 2 weeks after loading with articular chondrocytes yielded cartilaginous tissue formation selectively in the upper layer. Conversely, ectopic implantation in nude mice of the material after loading with bone marrow stromal cells resulted in bone formation which remained confined within the lower layer. In conclusion, we developed a composite material with cues which are biomimetic of an osteochondral tissue and with the capacity to differentially support cartilage and bone tissue generation. The results warrant testing of the material as a substitute for the repair of osteochondral lesions in orthotopic animal models.     

Preparation and histological evaluation of biomimetic three-dimensional hydroxyapatite/chitosan-gelatin network composite scaffolds

A novel biodegradable hydroxyapatite/chitosan-gelatin network (HA/CS-Gel) composite of similar composition to that of normal human bone was prepared as a three-dimensional biomimetic scaffold by phase separation method for bone tissue engineering. Changing the solid content and the compositional variables of the original mixtures allowed control of the porosities and densities of the scaffolds. The HA granules were dispersed uniformly in the organic network with intimate interface contact via pulverizing and ultrasonically treating commercial available HA particles. Scaffolds of 90.6% porosity were used to examine the proliferation and functions of the cells in this three-dimensional microenvironment by culturing neonatal rat caldaria osteoblasts. Histological and immunohistochemical staining and scanning electron microscopy observation indicated that the osteoblasts attached to and proliferated on the scaffolds. Extracellular matrices including collagen I and proteoglycan-like substrate were synthesized, while osteoid and bone-like tissue formed during the culture period. Furthermore, the cell/scaffold constructs had good biomineralization effect after 3 weeks in culture.     

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at -20 degrees C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140 degrees C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 microm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.     

Three-dimensional porous hydroxyapatite/collagen composite with rubber-like elasticity

A three-dimensional porous hydroxyapatite/collagen (HAp/Col) composite with a random pore structure was fabricated using freeze-drying processes; the self-organized HAp/Col nanocomposite with a weight ratio of 80.5:19.5, freeze-dried, was kneaded in 100 mM sodium phosphate buffer, frozen at ?20°C and freeze-dried. The cross-linkage of Col molecules was introduced dehydrothermally at 140°C in vacuo. The porous composite had a porosity of 94.7% with pore sizes between 200 and 500 μm. The compressive stress for the wet porous composite in phosphate buffer saline (PBS) was gradually decreased during 20 days incubation with a small amount of weight loss. The cyclic and time-course compression tests showed good repeatability of stress and well-recovery of its height, and caused no collapse of the porous composite. The implantation of the porous composite in rat bone holes showed the biodegradable property and new bone formation occurred in the pores without inflammatory response. The porous composite fabricated has good flexibility and rubber-like elasticity, and is a promising bone regenerative material.     

Incorporation of a decorin biomimetic enhances the mechanical properties of electrochemically aligned collagen threads

Orientational anisotropy of collagen molecules is integral to the mechanical strength of collagen-rich tissues. We have previously reported a novel methodology to synthesize highly oriented electrochemically aligned collagen (ELAC) threads with mechanical properties approaching those of native tendon. Decorin, a small leucine-rich proteoglycan (SLRP), binds to fibrillar collagen and has been suggested to enhance the mechanical properties of tendon. Based on the structure of natural decorin, we have previously designed and synthesized a peptidoglycan (DS-SILY) that mimics decorin both structurally and functionally. In this study, we investigated the effect of the incorporation of DS-SILY on the mechanical properties and structural organization of ELAC threads. The results indicated that the addition of DS-SILY at a molar ratio of 30:1 (collagen:DS-SILY) significantly enhanced the ultimate stress and ultimate strain of the ELAC threads. Furthermore, differential scanning calorimetry revealed that the addition of DS-SILY at a molar ratio of 30:1 resulted in a more thermally stable collagen structure. However, addition of DS-SILY at a higher concentration (10:1 collagen:DS-SILY) yielded weaker threads with mechanical properties comparable to collagen control threads. Transmission electron microscopy revealed that the addition of DS-SILY at a higher concentration (10:1) resulted in pronounced aggregation of collagen fibrils. More importantly, these aggregates were not aligned along the long axis of the ELAC, thereby compromising the overall tensile properties of the material. We conclude that incorporation of an optimal amount of DS-SILY is a promising approach to synthesize mechanically competent collagen-based biomaterials for tendon tissue engineering applications.     

Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering

In this study, we prepared nano-hydroxyapatite/polyamide (n-HA/PA) composite scaffolds utilizing thermally induced phase inversion processing technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Mesenchymal stem cells (MSCs) derived from bone marrow of neonatal rabbits were cultured, expanded and seeded on n-HA/PA scaffolds. The MSC/scaffold constructs were cultured for up to 7 days and the adhesion, proliferation and differentiation of MSCs into osteoblastic phenotype were determined using MTT assay, alkaline phosphatase (ALP) activity and collagen type I (COL I) immunohistochemical staining and scanning electronic microscopy (SEM). The results confirm that n-HA/PA scaffolds are biocompatible and have no negative effects on the MSCs in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure n-HA/PA scaffolds and MSC/scaffold constructs were implanted in rabbit mandibles and studied histologically and microradiographically. The results show that n-HA/PA composite scaffolds exhibit good biocompatibility and extensive osteoconductivity with host bone. Moreover, the introduction of MSCs to the scaffolds dramatically enhanced the efficiency of new bone formation, especially at the initial stage after implantation. In long term (more than 12 weeks implantation), however, the pure scaffolds show as good biocompatibility and osteogenesis as the hybrid ones. All these results indicate that the scaffolds fulfill the basic requirements of bone tissue engineering scaffold, and have the potential to be applied in orthopedic, reconstructive and maxillofacial surgery.     

The preparation and characteristics of a carbonated hydroxyapatite/collagen composite at room temperature

Nanocarbonated hydroxyapatite/collagen (nCHAC) composite was prepared at room temperature via biomimetic self-assembly method. X-ray diffraction (XRD), thermogravimetric analysis (TGA), and transmission electron microscopy (TEM) were performed. This composite shows the same inorganic phase of natural bone with nanosized level and low degree of crystallinity, and contains 2.8-14.7 wt % of carbonated content. TEM results confirm that the microstructure of this composite is the mineralized collagen fiber bundle like the hierarchical structure of natural bone. The diameter of a single mineralized collagen fiber is about 4 nm. Slightly different assembly units of the composite with different carbonates and collagen were demonstrated. The carbonated percentage affects the mineral crystal size and collagen fibril assembly. Because of the biomimetic component and microstructure, the use of nCHAC composite is promising for hard tissue therapy.     

Preparation of porous collagen/hyaluronic acid hybrid scaffolds for biomimetic functionalization through biochemical binding affinity

This study demonstrated the feasibility of introducing an avidin-biotin system to three-dimensional and highly porous scaffolds for the purpose of designing scaffolds that have binding affinity with bioactive molecules for various biomimetic modifications. Porous hybrid scaffolds composed of collagen and hyaluronic acid (HA) were prepared by a novel overrun process. The overrun-processed scaffolds showed a uniform dual-pore structure because of the injection of gas bubbles and ice recrystallization during the fabrication process and had a higher porosity than scaffolds prepared by a conventional freeze-drying method. The mechanical strength and biodegradation kinetics were controlled by the method of preparation and the composition of collagen/HA. Collagen/HA scaffolds did not show any significant adverse effects on cell viability even after 10 days of incubation. The fibroblasts cultured in the overrun-processed scaffolds were widely distributed and had proliferated on the surfaces of the macropores in the scaffolds, whereas the cells that were seeded in the freeze-dried scaffolds had attached mainly on the dense surface of the scaffolds. As the collagen content in the scaffolds increased, the cellular ingrowth into the inner pores of the scaffolds decreased because of the high affinity between the collagen and the cells. Measurements obtained via confocal microscopy revealed that the porous collagen/HA scaffolds could be functionalized with the biotin by incorporating avidin. Therefore, the present biotinylation approach may allow the incorporation of various bioactive molecules (DNA, growth factors, drug, peptide, etc) into the three-dimensional porous scaffolds.     

Electrospun PHBV/collagen composite nanofibrous scaffolds for tissue engineering

Electrospinning has recently emerged as a leading technique for the formation of nanofibrous structures made of synthetic and natural extracellular matrix components. In this study, nanofibrous scaffolds were obtained by electrospinning a combination of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV) and type-I collagen in 1,1,1,3,3,3-hexafluoro-2-isopropanol (HIFP). The resulting fibers ranged from 300 to 600 nm in diameter. Their surfaces were characterized by attenuated total reflection Fourier transform infrared spectroscopy, electron spectroscopy for chemical analysis and atomic force microscopy. The PHBV and collagen components of the PHBV/collagen nanofibrous scaffold were biodegraded by PHB depolymerase and a type-I collagenase aqueous solution, respectively. The cell culture experiments indicated that the PHBV/collagen nanofibrous scaffold accelerated the adhesion and growth of NIH3T3 cells more effectively than the PHBV nanofibrous scaffold, thus making the former a good scaffold for tissue engineering.     

Gradient collagen/nanohydroxyapatite composite scaffold: development and characterization

This paper reports an in situ diffusion method for the fabrication of compositionally graded collagen/nanohydroxyapatite (HA) composite scaffold. The method is diffusion based and causes the precipitation of nano-HA crystallites in situ. A collagen matrix acts as a template through which calcium ions (Ca(2+)) and phosphate ions (PO4(3-)) diffuse and precipitate a non-stoichiometric HA. It was observed that needle-like prismatic nano-HA crystallites (about 2 x 2 x 20 nm) precipitated in the interior of the collagen template onto the collagen fibrils. Chemical and microstructural analysis revealed a gradient of the Ca to P ratio across the width of the scaffold template, resulting in the formation of a Ca-rich side and a Ca-depleted side of scaffold. The Ca-rich side featured low porosity and agglomerates of the nano-HA crystallites, while the Ca-depleted side featured higher porosity and nano-HA crystallites integrated with collagen fibrils to form a porous network structure.     

Copper nanoparticle cues for biomimetic cellular assembly of crosslinked elastin fibers

Elastin, a structural protein distributed in the extracellular matrix of vascular tissues, is critical to maintaining the elastic stability and mechanical properties of blood vessels, as well as regulating cell-signaling pathways involved in vascular injury response and morphogenesis. Pathological degradation of vascular elastin or its malformation within native vessels and the poor ability to tissue-engineer elastin-rich vascular replacements due to innately poor elastin synthesis by adult vascular cells can compromise vascular homeostasis, and must thus be addressed. Our recent studies attest to the utility of hyaluronan (HA) oligomers for elastin synthesis and organization by adult vascular smooth muscle cells (SMCs), though the elastin matrix yields in these cases were quite low relative to total elastin produced. Thus, in this study, we investigated the utility of copper (Cu(2+)) ions to enhance cellular elastin deposition, crosslinking and maturation into structural fibers. Copper nanoparticles (CuNPs; 80-100 nm) in the dose range of 1-100 ng ml(-1) were tested for Cu(2+) ion release, and based on mathematical modeling of their release profiles, CuNPs (1, 10, and 400 ng ml(-1)) were chosen for supplementation to adult SMC cultures. The 400 ng ml(-1) dose of CuNPs cumulatively delivered Cu(2+) doses in the range of 0.1 M, over the 21 day culture period. It was observed that while exogenous CuNP supplements do not up-regulate tropoelastin production by vascular SMCs, they promoted formation of crosslinked elastin matrices. The deposition of crosslinked matrix elastin was further improved by the additional presence of HA oligomers in these cultures. Immunofluorescence imaging and structural analysis of the isolated elastin matrices indicate that amorphous elastin clumps were formed within non-additive control cultures, while aggregating elastin fibrils were observed within SMC cultures treated with CuNPs (1-10 ng ml(-1)) alone or together with HA oligomers. The presence of 400 ng ml(-1) of CuNPs concurrent with HA oligomers furthered aggregation of these elastin fibrils into mature fibers with diameters ranging from 200 to 500 nm. Ultrastructural analysis of elastin matrix within cultures treated with HA oligomers and 400 ng ml(-1) of CuNPs suggest that elastin matrix deposition as stimulated by Cu(2+) ions proceeds via a fibrillin-mediated assembly process, with enhanced crosslinking occurring via stimulation of lysyl oxidase. Overall, the data suggest that CuNPs and HA oligomers are highly useful for regenerating crosslinked, fibrillar elastin matrices by adult vascular SMCs. These results have immense utility in tissue-engineering vascular replacements.     

Development of an artificial vertebral body using a novel biomaterial,hydroxyapatite/collagen composite

Hydroxyapatite/collagen (HAp/Col) composites having a bone-like nanostructure were synthesized and shaped into implants. This study was designed to develop an artificial vertebra system using this novel implant for anterior fusion of the cervical spine. Anterior fusion was carried out on 6 beagle dogs with the implants adsorbing rhBMP-2 (400 microg/ml). and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate and 2 titanium screws. Implants were taken out after 13 weeks from each 3 dogs in the rhBMP(-):plate(-). rhBMP(-):plate(+) and rhBMP(+):plate(+) groups. Also, the implants were removed from each 3 dogs in the rhBMP(-):plate(+) and rhBMP(+):plate(+) groups after 24 weeks. Histological and radiographical analysis suggested that since the larger part of the composite material was absorbed within 13 weeks, reduction of the intervertebral distance was caused, and that enhancement of callus formation and bone bridging by rhBMP-treatment was effective to prevent collapse of the implant, even though an effect of anterior plate-fixation was not obvious. The HAp/Col implant adsorbing rhBMP-2 may be a suitable replacement for the existing ceramics in anterior interbody fusion of the cervical spine.     

Development of a novel biomaterial, hydroxyapatite/collagen (HAp/Col) composite for medical use

A hydroxyapatite/type I collagen (HAp/Col) composite, aligning hydroxyapatite nano-crystals along collagen molecules, has been synthesized. The biocompatibility, osteoconductivity and efficacy as an rhBMP-2 carrier of this novel biomaterial implanted in the weight-bearing site have been examined. The HAp/Col implants adsorbing 0 or 400 microg/ml of rhBMP-2 were implanted into bone defects of tibiae in 3 beagle dogs and fixed according to the Ilizarov method. As a control, bone defects of 20 mm remaining in 2 beagle dogs and the dogs were allowed to walk using a Ilizarov external skeletal fixator. The radiological and histological findings suggest that the implants induce bone remodeling units and are a superior carrier of rhBMP-2 due to the stimulation of early callus and new bone formation. As a next step, anterior fusion was carried out on 6 beagle dogs with the implants adsorbing 400 microg/ml of rhBMP-2, and 9 dogs with the implants without rhBMP-2. In 3 dogs of the rhBMP-treated group, as well as 6 dogs of the non-rhBMP-treated group, the implant was fixed with a poly-L-lactide plate. Histological and radiographical analysis suggest that enhancement of callus formation and bone bridging by rhBMP-treatment is effective to prevent collapse of the implant.     

In vivo evaluation and comparison of collagen, acetylated collagen and collagen/glycosaminoglycan composite films and sponges as candidate biomaterials

Native collagen, acetylated collagen, collagen/10% chondroitin sulphate, collagen/2.5% hyaluronic acid and collagen/20% hyaluronic acid were implanted both as film and as sponge into rat lumbar muscle for 7 and 14 d. After 7 d implantation, all materials elicited an acute inflammatory cell response characterized by numerous polymorphs and histocytes. The cell population after 14 d was principally mononuclear, i.e. leucocytes, neutrophils, macrophages, lymphocytes and fibroblasts. Both films and sponges followed a similar pattern. Native collagen elicited a subacute inflammatory response after 7 d. However, 14 d after implantation, a marked infiltration by neutrophils was apparent with subsequent degradation of existing collagen material. Acetylated collagen film evoked a much greater inflammatory cell response than native collagen. Both collagen/hyaluronic acid composites elicited a similar response. The collagen/10% chondroitin sulphate composite elicited the least inflammatory cell response at 7 d, whereas infiltration by host fibroblasts after 14 d implantation was clearly seen.