Effect of chondroitin sulphate on material properties and bone remodelling around hydroxyapatite/collagen composites

Chondroitin sulphate (CS) has an anti-inflammatory effect and increases the regeneration ability of injured bone. The goal of this study was to characterize the material properties and osteoconductive potency of calcium phosphate bone cements modified with CS. The early interface reaction of cancellous bone to a nanokristalline hydroxyapatite cement containing type I collagen (HA/Coll) without and with CS (HA/Coll/CS) in a rat tibia model was evaluated. Cylindrical implants were inserted press-fit into defect of the tibial head. Six specimens per group were analyzed at 2, 4, 7, 14, and 28 days. HA/Coll/CS composite cylinders showed a 15% increase in compressive strength and by investigations with powder X-ray diffraction more nontransformed cement precursor was found. The microstructures of both types of implants were similar. A significantly higher average number of TRAP positive osteoclasts and ED1 positive mononuclear cells were observed in the interface around HA/Coll/CS implants on day 4 and 7 (p < 0.05). At 28 days the direct bone contact and the percentage of newly formed bone were significantly higher around HA/Coll/CS implants (p < 0.05). The addition of CS appears to enhance bone remodelling and new bone formation around HA/Coll composites in the early stages of bone healing. Possible mechanisms are discussed.     

胶原/羟基磷灰石复合材料的制备及用于骨缺损修复的研究现状

通过模拟天然骨的结构,制备胶原/羟基磷灰石复合材料,与天然骨具有相似的组成、结构和性能,并具有良好的生物活性和生物降解性。本文就胶原/羟基磷灰石复合材料的制备方法、仿生形成机制、表征手段及骨缺损修复的应用等进行综述,并展望其未来发展方向。     

Quick-forming hydroxyapatite/agarose gel composites induce bone regeneration

We investigated the fundamental properties of quick-forming hydroxyapatite (HAp)/agarose gel composites, and evaluated their potential as an injectable bone substitute. From scanning electron microscope observations, the HAp/agarose gel composites produced by an innovative electrophoretic process showed an interconnecting structure with the HAp particles. The diameter of the HAp particles was roughly 1 microm, and the total amount of HAp particles was estimated by a quantification of the calcium ions. In the case of 1 mg of dry composite, 10 microg of HAp was formed in the agarose gel. Moreover, X-ray diffraction analysis revealed that the HAp particles had an amorphous structure, so the HAp particles were expected to dissolve under physiological conditions relative to the HAp with higher crystallinity. The advantages of the resultant HAp/agarose gel composites are ease of handling, close contact with the surrounding tissues, and ease of use as an injectable material. As a preliminary animal study, the composites were implanted into the medial femoral condyle of rabbits. After implantation, the process of bone regeneration was evaluated by microfocus-computed tomography (microCT) and histological analysis. At 2 weeks postoperatively, newly-formed bone was observed at the edge of the bone defect site, and at 4 weeks postoperatively, excellent bone regeneration was observed. The implanted composite gradually degraded, and disappeared at 8 weeks postoperatively. This result indicated that the composite dissolved rapidly, and was replaced by newly-formed bone. Quick-forming HAp/agarose gel composites may be a good candidate as an injectable biomaterial, particularly in the fields of orthopedic, oral, and maxillofacial surgery.     

Three-layered membranes of collagen/hydroxyapatite and chitosan for guided bone regeneration

The purpose of this study was to design a novel hybrid membrane with optimized properties for guided bone regeneration (GBR). Both the top and bottom layers of the sandwich-structured membrane were composed of collagen containing 20 wt% hydroxyapatite (HA), while the middle layer was made of chitosan. The above three layers were formulated into an integral membrane from their respective slurries through a layer-by-layer filtration process. The phase and composition of the membrane were confirmed by FT-IR and XRD analyses. The observation of its morphology by SEM showed that the membrane had a porous structure and structural integrity. The chitosan layer ensured the high tensile strength and elastic modulus of the membrane, while the presence of the collagen/HA composite layers endowed it with good flexibility and bioactivity. These results suggest that the integrated membrane prepared in this study would have the potential for use as a GBR material.     

羟基磷灰石／胶原人工骨在创伤后骨缺损修复中的初步研究

目的评估羟基磷灰石，胶原（hydroxyapatite/collagen，HAC）人工骨在创伤后骨缺损修复中的临床效果。方法48例创伤后四肢骨骨折患者，无其他病史，分为2组。试验组25例，其中男性15例，女性10例，年龄22～82岁，平均年龄45.3岁。对照组患者23例，其中男性13例，女性10例，年龄21～81岁，平均年龄46岁。对所有患者进行骨折切开复位，选择合适的内固定。试验组于固定后，在骨缺损处、骨髓腔、骨折线周围直接植骨，羟基磷灰石，胶原人工骨修复材料植入量0.4～4.0g不等。对照组不植入羟基磷灰石，胶原人工骨。结果两组46例病例获随访。试验组中2例失随访，余23例患者无局部或全身不良反应，骨折均达临床愈合。X线片显示,术后1～3个月材料植入区与缺损周围的骨组织间界限模糊，有新骨形成，3～6个月材料植入区内有明显的新骨长入，人工骨与骨组织融合，骨缺损基本修复。对照组3例延迟愈合和1例骨折不愈合患者在功能上恢复较差；试验组与对照组比较具有统计学意义，P〈0．05。结论胶原，羟基磷灰石人工骨具有良好的材料，细胞界面，使材料本身更具备与骨键合的能力。其三维多孔结构便于骨组织的长入，生物降解性有利于骨组织的改建和塑形，具有良好的临床应用前景。     

Proteinoid/hydroxyapatite hybrid microsphere composites

Organic/hydroxyapatite (HA) hybrid composites are promising materials for orthopedic applications. Proteinoid/HA hybrid microsphere composite is an ideal material for bioresorbable and biocompatible three-dimensional scaffolds. Proteinoids are thermally condensed mixtures of amino acids, forming microspheres via self-assembly of proteinoid chains. Synthesis of an onion-type multilayer proteinoid/HA hybrid composite is achieved by thermally condensing a mixture of amino acids in the presence of commercially available HA. Proteinoid/HA microsphere composite of about 40 μm are achieved as a result of heterogeneous nucleation of HA on proteinoids chains. The formation mechanism is based on the interaction between calcium ions of HA and carboxyl side groups of proteinoid. The morphology was gleaned by scanning electron microscopy, and the organic and inorganic constituents were characterized by several analytical methods.     

Glutaraldehyde cross-linked hydroxyapatite/collagen self-organized nanocomposites

To control the mechanical properties and biodegradability of self-organized hydroxyapatite/collagen (HAp/Col) nanocomposites, cross-linkage was introduced into the composites with glutaraldehyde (GA). The HAp/Col composite suspensions, prepared by a simultaneous titration method and aged for 3h, were cross-linked with the reagents for 10min under vigorous stirring. The precipitates obtained were filtrated and compacted by dehydration under a uniaxial pressure. The particle size distribution, 3-point bending strength, contained water amount and swelling ratio of the composites were examined as a function of cross-linkage amount; the biodegradability was estimated by animal tests using rabbits. As regards the cross-linked composites, no long-rage alignment of HAp crystals along collagen molecules was found with a transmission electron microscope, suggesting that the cross-linking reagents suppressed their long-range self-organization mechanism. The 3-point bending strength increased with the GA content and took a maximal value at 1.35mmol/g(col). The animal tests indicated no toxicity and osteoclastic resorption with good osteoconductivity. The resorption rate was decreased with increasing GA concentration. These results suggest that GA cross-linkage controls mechanical properties and resorption rate without reducing high biocompatibility of the composite.     

Octacalcium phosphate combined with collagen orthotopically enhances bone regeneration

Octacalcium phosphate (OCP) is resorbable bone regenerative material, but its brittleness makes it difficult to maintain its shape without restraint. We have engineered a scaffold constructed of synthetic OCP and porcine collagen sponge (OCP/Collagen) and investigated whether OCP/Collagen composite could improve bone regeneration. To examine this hypothesis, bone regeneration by the implantation of OCP/Collagen was compared with those by OCP and collagen. Radiographic and histological examination was performed and the percentage of newly formed bone (n-Bone%) in the defect was determined by a histomorphometrical analysis. OCP/Collagen, OCP, or collagen was implanted into the critical-sized defects in rat crania and fixed at 2, 4, or 8 weeks after implantation. OCP/Collagen improved the handling performance than the granules of OCP, and synergistically enhanced the bone regeneration beyond expectation, which were composed of bone nucleation by OCP and cell infiltration by collagen. Histomorphometrical analysis showed that n-Bone% +/- standard error treated with OCP/Collagen (48.4 +/- 5.14) was significantly higher than those with OCP (27.6 +/- 4.04) or collagen (27.4 +/- 5.69) in week 8. The present study suggests that the combination OCP with collagen elicited the synergistic effect for bone regeneration.     

纳米相羟基磷灰石/胶原复合材料研究进展

综述羟基磷灰石(hydroxyapatite,HA)/胶原复合材料的研究进展,着重阐述自组装纳米相羟基磷灰石/胶原复合材料的制作方法、结构特点、体内植入后修复骨缺损的效果及降解过程。基于仿生学设计的纳米相羟基磷灰石/胶原复合材料,其HA纳米晶体约50～100nm,HA的C-轴沿胶原纤维排列,形成片状包绕胶原纤维束,HA和胶原分子之间为牢固的化学键性结合,为自组装的纳米结构,和自然骨中钙化的胶原相同。复合材料体内植入后降解和骨替代的过程与骨的改建过程相似。纳米相羟基磷灰石/胶原复合材料具有生物降解性高、表面能大、生物活性好、生物相容性好等特点,作为骨修复和重建材料具有更好的前景。     

纳米相羟基磷灰石/胶原复合材料研究进展

综述羟基磷灰石（hydroxyapatite,HA）/胶原复合材料的研究进展,着重阐述自组装纳米相羟基磷灰石/胶原复合材料的制作方法、结构特点、体内植入后修复骨缺损的效果及降解过程.基于仿生学设计的纳米相羟基磷灰石/胶原复合材料,其HA纳米晶体约50～100nm,HA的C-轴沿胶原纤维排列,形成片状包绕胶原纤维束,HA和胶原分子之间为牢固的化学键性结合,为自组装的纳米结构,和自然骨中钙化的胶原相同.复合材料体内植入后降解和骨替代的过程与骨的改建过程相似.纳米相羟基磷灰石/胶原复合材料具有生物降解性高、表面能大、生物活性好、生物相容性好等特点,作为骨修复和重建材料具有更好的前景.     

In vitro analysis of biodegradable polymer blend/hydroxyapatite composites for bone tissue engineering.

Blends of biodegradable polymers, poly(caprolactone) and poly(D, L-lactic-co-glycolic acid), have been examined as scaffolds for applications in bone tissue engineering. Hydroxyapatite granules have been incorporated into the blends and porous discs were prepared. Mechanical properties and degradation rates in vitro of the composites were determined. The discs were seeded with rabbit bone marrow or cultured bone marrow stromal cells and incubated under physiological conditions. Polymer/ceramic scaffolds supported cell growth throughout the scaffold for 8 weeks. Scanning and transmission electron microscopy, and histological analyses were used to characterize the seeded composites. This study suggests the feasibility of using novel polymer/ceramic composites as scaffold in bone tissue engineering applications.     

Poly-epsilon-caprolactone/hydroxyapatite composites for bone regeneration: in vitro characterization and human osteoblast response

Polycaprolactone (PCL), a semicrystalline linear resorbable aliphatic polyester, is a good candidate as a scaffold for bone tissue engineering, due to its biocompatibility and biodegradability. However, the poor mechanical properties of PCL impair its use as scaffold for hard tissue regeneration, unless mechanical reinforcement is provided. To enhance mechanical properties and promote osteoconductivity, hydroxyapatite (HA) particles were added to the PCL matrix: three PCL-based composites with different volume ratio of HA (13%, 20%, and 32%) were studied. Mechanical properties and structure were analysed, along with biocompatibility and osteoconductivity. The addition of HA particles (in particular in the range of 20% and 32%) led to a significant improvement in mechanical performance (e.g., elastic modulus) of scaffold. Saos-2 cells and osteoblasts from human trabecular bone (hOB) retrieved during total hip replacement surgery were seeded onto 3D PCL samples for 1-4 weeks. Following the assessment of cell viability, proliferation, morphology, and ALP release, HA-loaded PCL was found to improve osteoconduction compared to the PCL alone. The results indicated that PCL represents a potential candidate as an efficient substrate for bone substitution through an accurate balance between structural/ mechanical properties of polymer and biological activities.     

Development of hydroxyapatite/calcium silicate composites addressed to the design of load-bearing bone scaffolds

This work deals with the preparation of bioactive ceramic composites to be employed for the development of load-bearing bone substitutes, made of hydroxyapatite (Ca₁₀(PO₄)₆(OH)₂, HA) and bioactive dicalcium silicate (Ca₂SiO₄, C₂S) as a reinforcing phase. The composite materials were prepared by Fast Hot-Pressing (FHP), which allowed the rapid sintering of monolithic ceramics at temperatures up to 1500 ^°C, well above the commonly adopted temperatures for the consolidation of hydroxyapatite (1200–1300 ^°C). The purpose was to achieve the grain coalescence of both HA and the strengthening phase, so that to obtain a homogeneous ceramic material characterized by controlled phase composition and improved mechanical strength; the dwell time was reduced as much as possible to prevent HA decomposition and excessive grain growth. The most remarkable result, in terms of phase composition, was the absence of any secondary phases in the final ceramics other than HA and C₂S, even after sintering at 1500 ^°C. The flexure strength of the composite materials was found to be much higher than that of HA alone. Further mechanical characterization was also carried out on HA and composites, sintered in different conditions, to evaluate the elastic properties and fracture toughness, and properties close to those of mineral bone were found. These preliminary results confirmed that composites of HA and Ca₂SiO₄ are promising for the development of bioactive load-bearing ceramic bone substitutes with controlled phase composition.     

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。     

羟基磷灰石/聚乳酸复合人工骨修复材料的研究进展

近年来,羟基磷灰石/聚乳酸复合生物材料用做人工骨修复材料得到了广泛的研究。本文综述了近年来羟基磷灰石/聚乳酸(HA/PLA)复合人工骨修复材料的各种制备方法及其性能影响因素的研究进展。     

BMP-2 incorporated in a tricalcium phosphate bone substitute enhances bone remodeling in sheep

Bone morphogenetic protein-2 (BMP-2) is a well-known osteoinductive protein, which requires a carrier for local application. As an alternative to the previously described carriers, an in situ hardening, resorbable, and osteoconductive beta-tricalcium phosphate cement (TCP) is tested. Trepanation defects in the bovine distal femoral epiphysis are filled with a composite consisting of TCP and 200 microg rhBMP-2 per cm3 TCP, autologous bone graft, pure TCP, or left empty. A radiological follow-up is performed after 7 weeks and 3 months. The sheep are euthanized and bone samples are analyzed by microradiography, histology, and histomorphometry. Microradiography and histology show similar results for pure TCP and the composite. The defects are filled with trabecular bone and newly formed bone is in close contact with the remaining TCP-particles. The majority of the cement is resorbed, in the composite group the amount of remaining cement particles is reduced. Defects treated with autologous bone graft are filled completely, while untreated defects shows only a small amount of bone originating from the rim of the defect. Histomorphometry of the defects treated with pure TCP shows a significantly increased bone content in comparison to defects treated with the composite or autologous bone graft. Analysis of the remaining cement particles shows significantly less cement in the TCP/rhBMP-2 group in comparison to pure TCP. The sum of bone and cement content in the rhBMP-2 group shows amounts comparable to the calcified structures found following autologous bone grafting. The addition of rhBMP-2 to the TCP leads to faster remodeling of the defect comparable to autologous bone graft, while defects treated with pure TCP are not completely remodeled.     

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.     

The primacy of octacalcium phosphate collagen composites in bone regeneration

We have engineered a scaffold constructed of synthetic octacalcium phosphate (OCP) and porcine collagen sponge (OCP/Col), and reported that OCP/Col drastically enhanced bone regeneration. In this study, we investigated whether OCP/Col would enhance bone regeneration more than beta-tricalcium phosphate (beta-TCP) collagen composite (beta-TCP/Col) or hydroxyapatite (HA) collagen composite (HA/Col). Discs of OCP/Col, beta-TCP/Col, or HA/Col were implanted into critical-sized defects in rat crania and fixed at 4 or 12 weeks after implantation. The newly formed bone and the remaining granules of implants in the defect were determined by histomorphometrical analysis, and radiographic and histological examinations were performed. Statistical analysis showed that the newly formed bone by the implantation of OCP/Col was significantly more than that of beta-TCP/Col or HA/Col. In contrast, the remaining granules in OCP/Col were significantly lower than those in beta-TCP/Col or HA/Col. Bone regeneration by OCP/Col was based on secured calcified collagen and bone nucleation by OCP, whereas bone regeneration by beta-TCP/Col or HA/Col was initiated by poorly calcified collagen and osteoconductivity by beta-TCP or HA. This study showed that the implantation of OCP/Col in a rat cranial defect enhanced more bone regeneration than beta-TCP/Col and HA/Col.