Biologically inspired synthesis of bone-like composite: self-assembled collagen fibers/hydroxyapatite nanocrystals

Replacement of bone tissue by graft materials and products of tissue engineering having composition, structure, and biological features that mimic natural tissue is a goal to be pursued. A biomimetic synthesis was performed to prepare new bone-like composites constituted of hydroxyapatite nanocrystals and self-assembled type I collagen fibers. We used a biological inspired approach that proved that the biological systems stored and processed information at the molecular level. Two different methodologies were used: dispersion of synthetic hydroxyapatite in telopeptides free collagen molecules solution and direct nucleation of hydroxyapatite into reconstituted collagen fibers during their assembling. The different preparation techniques were experimented then the composites thoroughly characterized and compared. Composite obtained by direct nucleation showed an intimated interaction of the inorganic and proteic components, which modified the apatitic phase and made its composition, morphology and structure similar to the mineral component of natural bone.     

FT-IR study for hydroxyapatite/collagen nanocomposite cross-linked by glutaraldehyde

FT-IR analysis was performed for the hydroxyapatite (HAp)/collagen (COL) nanocomposite cross-linked by glutaraldehyde (GA). The amide bands I, II and III from COL matrix, and phosphate and carbonate bands from HAp were identified. The amide B band arising from C–H stretching mode showed a sensitive conformation by the degree of cross-linking. The amide I band showed a complicate conformational change by the degree of cross-linking. The characteristic amide I band at 1685 cm⁻¹, which is known as an aging parameter in the biological bone, did not show a monotonous tendency by the degree of cross-linking. The relative contents of the organics in the cross-linked HAp/COL nanocomposite were evaluated as an integration ratio between the amide I band at 1600–1700 cm⁻¹ and PO₄³⁻ band at 900–1200 cm⁻¹. The increase of the organics content by the cross-linking is enabled by the further organization of Ca²⁺ ions of HAp crystals in HAp/COL nanocomposite. The complicate conformational behavior in the amide I, II and III bands seems to be affected by the cross-linking induced directional arrangement of HAp/COL nanocomposite fibrils.     

In vitro evaluation of nanosized carbonate-substituted hydroxyapatite and its polyhydroxyethylmethacrylate nanocomposite

Nanometer scale carbonate-substituted hydroxyapatite (nanoCHA) particles were prepared and examined using transmission electron microscopy, which revealed their polycrystalline nature with a rod-like morphology (20-30 nm in width and 50-80 nm in length). In vitro cytotoxicity study showed that there was some evidence of lactate dehydrogenase (LDH) release when macrophages were in contact with high concentrations of nanoCHA particles. The levels of LDH release decreased significantly with a reduction in nanoCHA concentration. A similar trend was observed for the inflammatory cytokine TNF-alpha. nanoCHA particles with high carbonate content induced a high level of TNF-alpha release. Biological testing using a human osteoblast (HOB) cell model found that HOB cells were able to grow and proliferate on a nanoCHA deposited surface. Well organized actin fibers were observed for HOB cells in contact with nanoCHA particles with low carbonate content and the cell proliferation rate was higher on these particles in comparison with those of high carbonate nanoCHA particles. Therefore, low carbonate nanoCHA particles were incorporated into poly-(2-hydroxyethylmethacrylate) matrix to make a nanocomposite. It was found that the nanoCHA composite was hydrophilic and became rubber-like after hydration. Both 20 wt % and 40 wt % composites were able to induce the formation of bone-like apatite after immersion in simulated body fluid. A high bioactivity of the composite was obtained with high loading of the nanoCHA filler. These results demonstrate the potential of formulating nanocomposites for biomedical applications.     

Effects of hydroxyapatite on endothelial network formation in collagen/fibrin composite hydrogels in vitro and in vivo

Co-culture of endothelial cells (EC) and mesenchymal stem cells (MSC) results in robust vascular network formation in constrained 3-D collagen/fibrin (COL/FIB) composite hydrogels. However, the ability to form endothelial networks is lost when such gels are allowed to compact via cell-mediated remodeling. In this study, we created co-cultures of human EC and human MSC in both constrained and unconstrained COL/FIB matrices and systematically added nanoparticulate hydroxyapatite (HA, 0–20 mg ml⁻¹), a bone-like mineral that has been shown to have pro-vasculogenic effects. Constructs cultured for 7 days were assayed for gel compaction, vascular network formation, and mechanical properties. In vitro, robust endothelial network formation was observed in constrained COL/FIB constructs without HA, but this response was significantly inhibited by addition of 5, 10, or 20 mg ml⁻¹ HA. In unconstrained matrices, network formation was abolished in pure COL/FIB constructs but was rescued by 1.25 or 2.5 mg ml⁻¹ HA, while higher levels again inhibited vasculogenesis. HA inhibited gel compaction in a dose-dependent manner, which was not correlated to endothelial network formation. HA affected initial stiffness of the gels, but gel remodeling abrogated this effect. Subcutaneous implantation of COL/FIB with 0, 2.5 or 20 mg ml⁻¹ HA in the mouse resulted in increased perfusion at the implant site, with no significant differences between materials. Histology at day 7 showed both host and human CD31-stained vasculature infiltrating the implants. These findings are relevant to the design of materials and scaffolds for orthopedic tissue engineering, where both vasculogenesis and formation of a mineral phase are required for regeneration.     

In vitro and in vivo biological responses of plasma-sprayed hydroxyapatite coatings with posthydrothermal treatment

This study was undertaken to evaluate the effect of post-hydrothermal treatment on the biological responses of the plasma-sprayed hydroxyapatite (HA)-coated Ti-6Al-4V implant system both in vitro and in vivo. After hydrothermal treatment, the HA coating (HAC) shows the high mechanical strength and indices-of-crystallinity, denser microstructure, lower concentrations of amorphous and impurity phases, when compared with the as-sprayed HAC. The in vitro cell-culture studies, using UMR106 osteoblast-like cell, demonstrated no signifiacnt cell growth on both surface of as-sprayed and hydrothermal-treated HACs during 10-day culture. The in vivo studies, using the transcortical implant model in the femora of goats, evaluated the histological responses of two coatings. After 6 week of implantation, using backscattered electron images, no substantial histological variations in the extents of new bone apposition and new bone healing between the two HACs were observed. However, the as-sprayed HAC, owing to the dissolution induced the granular particles dissociated from the HAC, showed the statically lower extent of new bone apposition than hydrothermal-treated HAC at 12 weeks. The results suggest that hydrothermal treatment could be used to improve the mechanical strength, crystallinity, and phase composition of HAC, which are important factors of long-term fixation and stability of implant. Besides, the treated HAC could also achieve the initial fixation of implant in clinical use.     

Preparation of a collagen/polymer hybrid gel for tissue membranes. Part II: In vitro and in vivo biological properties of the collagen gels

The aim of the present study was to evaluate the biological properties of a collagen–phospholipid polymer hybrid gel (MiC30 gel) designed for use as a tissue membrane. The following four types of collagen gels were synthesized and tested in vitro and in vivo: physically cross-linked collagen gel (Uc gel), N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC)/N-hydroxysuccinimide-cross-linked collagen gel (EN gel), MiC30 gel and glutaraldehyde-cross-linked collagen gel (G gel). The cell adhesivity and proliferation rate were observed to be lowest for the MiC30 gel and highest for the Uc gel, indicating that the phospholipid–polymer-covered surface of the collagen gel interacted weakly with cells. The collagen gel was implanted into rats subcutaneously and was observed 1, 2 and 8 weeks after implantation. The Uc gel and G gel were degraded and induced an inflammatory response. Granulation was not observed for 8 weeks after implantation and the formation of foreign body giant cells was observed around both the Uc and G gels. On the other hand, cell infiltration and degradation were not observed in the case of the EN and MiC30 gels. The formation of foreign body giant cells was suppressed and the healing process was accelerated. The MiC30 gel is suitable for use as a biomaterial that is stable in vivo because it suppresses the foreign body response and accelerates the healing process.     

Collagen-infiltrated porous hydroxyapatite coating and its osteogenic properties: in vitro and in vivo study

Bone-implant interface is critical for the early fixation of orthopedic implants. In this study, porous hydroxyapatite (HA) coatings were prepared through a liquid precursor plasma spraying process and were infiltrated with the collagen, alone and with the additional incorporation of recombinant human bone morphogenetic protein-2 (rhBMP-2) and RGD peptide (RGD). The results showed significantly improved mesenchymal stem cell (MSC) adhesion, proliferation, and differentiation on collagen-modified HA coatings, partially benefited from the formation of a fibrous network due to the self-reconstitution of collagen on the HA surface. Further enhancements on MSC proliferation and differentiation were generally observed through the additional incorporation of bone morphogenetic protein (BMP) and RGD. The osteoinductive and osteoconductive properties of the collagen/BMP-modified HA coatings were studied in vivo. Clear ectopic bone formation and significantly accelerated bone growth rate (29% increase, p < 0.05) have been observed after 1-month implantation of HA-collagen/rhBMP-2-coated Ti alloy samples into the rabbit muscle and dog femora, respectively. Overall, our results suggest that collagen-modified HA coating surface is a far superior substrate for cell attachment, proliferation, and differentiation, and collagen can be used an efficient carrier for BMP in vivo. Therefore, modification of HA coating with collagen is a simple but effective biomimetic approach to enhancing the osteointegration and early fixation of bone-implant interface.     

Synthesis and characterization of wool keratin/hydroxyapatite nanocomposite

Taking the inspiration from the biomineral, the wool keratin was selected to modulate the assembly of nanosized hydroxyapatite (HA) crystals via a coprecipitation method. A series of keratin/HA nanocomposite with different ratios were synthesized by adjusting the concentrations of keratin solutions and calcium phosphate and their final components were detected by thermogravimetric analysis (TGA). The transmission electron microscopy (TEM) and X-ray diffraction (XRD) confirmed that keratin in the composite decreased the crystallinity of HA. Fourier transform infrared (FTIR) and X-ray photoelectron spectroscopy (XPS) were used to examine the chemical and surface structure of the composites. In vitro biocompatibility results revealed that cells showed better viability on keratin/HA composites which have a ratio of organics and inorganics similar to that of natural bones.     

Mechanism of bone-like formation on a bioactive implant in vivo

The physical and chemical nature of the remodelled interface between the porous A3 glass-ceramic, composed of (wt%): SiO(2) = 54.5; CaO = 15.0; Na(2)O = 12.0; MgO = 8.5; P(2)O(5) = 6.0 K(2)O = 4.0, and the surrounding bone was studied after implantation into rat tibias. The interfaces which developed new bone layer in direct contact with the implants were examined by analytical scanning and transmission electron microscopy after implantation for 6, 8 and 12 weeks. Degradation processes of the implants also encouraged osseous tissue ingrowths into the pores of the material, changing drastically the macro- and microstructure of the implants. The ionic exchange initiated at the implant interface with the physiological environment was essential in the integration process of the implant, through a dissolution-precipitation-transformation mechanism. The interfaces developed non-toxic biological and chemical activities and remained reactive over the 12-week implantation period. These findings were significant as indicative of morphological and chemical integration of the A3 glass-ceramic into the structure of living bone tissue. A3 glass-ceramic could be suitable for the repair or replacement of living bone.     

Dynamic mechanical properties of a biomimetic hydroxyapatite/polyamide 6,9 nanocomposite

A biomimetic composite of nanohydroxyapatite (nHap) and semicrystalline polyamide 6,9 (PA 6,9) was synthesized by thermally induced phase separation. The nHap powder was dispersed in a polymer matrix with a low ratio ranging 1-10 wt %. The mean size of the nHap, determined by scanning electron microscopy (SEM) was approximately 100-200 nm (length), 40-60 nm (width). Physicochemical analyses were performed in order to characterize the PA 6,9 and nHap separately on the one hand, and the PA 6,9/nHap composites on the other hand. Differential scanning calorimetry (DSC) and dynamic mechanical analyses (DMA) have pointed out an optimization of the composite physical properties as a function of nHap content till a limit value of 5 wt %. Above this value, the mechanical properties decreased. Four main parameters have been found to influence the composite physical properties improvement: the fillers content, the physical structure of the polymeric matrix, the particles dispersion and the physical interaction strength between organic and inorganic phases. The dynamic mechanical properties of this biomimetic nanocomposite were compared with human cortical bone.     

Poly(vinyl alcohol)/collagen/hydroxyapatite hydrogel: Properties and in vitro cellular response

The objective of this study was to develop "bone-like" poly(vinyl alcohol) (PVA)/hydroxyapatite (HA)/type I collagen (Col) hydrogel composites that stimulate adhesion, proliferation, and differentiation of osteoblastic cells. The hydrogel composites were prepared by mixing PVA with nanoscale HA and Col using a physical mixing method. The concentration of the components was optimized during formulation development. PVA/Col/HA hydrogels were characterized for viscoelasticity, degree of swelling, mechanical strength, embedded erythromycin drug release, and cellular response of both osteoblastic MC3T3 cells and RAW 264.7 macrophage cells. Compressive strength tests confirmed that the PVA coating possessed greater elasticity and was mechanically enhanced by the freeze-thaw treatment. PVA/Col/HA gel is biocompatible and nontoxic to MC3T3 preosteoblasts, and the reinforcement from HA and Col reduced the inflammatory response from macrophages. Our findings demonstrate that PVA composites are biocompatible, and enhance cell adhesion, proliferation, and differentiation in vitro. We propose that PVA/Col/HA hydrogels represent one of the promising implant surface coating matrices for the improvement of implant osseointegration. ? 2012 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 100A:3071-3079, 2012.     

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.     

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。     

Ultrastructural comparison of dissolution and apatite precipitation on hydroxyapatite and silicon-substituted hydroxyapatite in vitro and in vivo

Recent histological studies have demonstrated that the substitution of silicate ions into hydroxyapatite (HA) significantly increases the rate of bone apposition to HA implants. The enhanced bioactivity of silicon-substituted HA (Si-HA) over pure HA has been attributed to the effect of silicate ions in accelerating dissolution. In the present study, high-resolution transmission electron microscopy (HR-TEM) was employed to compare dissolution of HA and Si-HA in an acellular simulated body fluid (SBF) to dissolution in an in vivo model. HR-TEM observations confirmed a difference in morphology of apatite precipitates in vivo and in SBF: apatite deposits were platelike in vivo and nodular in SBF. Compositional mapping suggested that preferential dissolution of silicon from the implant promotes the nucleation of carbonate apatite around the implant. The in vivo findings illustrated an absence of dissolution at the bone-HA or Si-HA interface, whereas dissolution was extensive from within the implant. The amount of dissolution in acellular SBF was similar to dissolution from within the implant, although the site at which the dissolution nucleates was different: dissolution predominates at the crystallite surfaces in SBF, whereas grain boundary dissolution predominates in vivo. These findings suggest that proteins in the in vivo milieu modify the processes of dissolution from the implant.     

Strontium-substituted hydroxyapatite coatings synthesized by pulsed-laser deposition: in vitro osteoblast and osteoclast response

The increasing interest in strontium incorporation into biomaterials for hard tissue repair is justified by the growing evidence of its beneficial effect on bone. We successfully synthesized hydroxyapatite (HA) thin films with different extents of strontium substitution for calcium (0, 1, 3 or 7 at.%) by pulsed-laser deposition. The coatings displayed a granular surface and a good degree of crystallinity, which slightly diminished as strontium content increased. Osteoblast-like MG63 cells and human osteoclasts were cultured on the thin films up to 21 days. MG63 cells grown on the strontium-doped HA coatings displayed normal morphology, good proliferation and increased values of the differentiation parameters, whereas the number of osteoclasts was negatively influenced by the presence of strontium. The positive effect of the ion on bone cells was particularly evident in the case of coatings deposited from HA at relatively high strontium contents (3-7%), where significantly increased values of alkaline phosphatase activity, osteocalcin, type I collagen and osteoprotegerin/TNF-related activation-induced cytokine receptor ratio, and considerably reduced values of osteoclast proliferation, were observed.     

Degradation of collagen suture in vitro and in vivo.

In vitro and in vivo degradation of collagen suture was investigated focussing on the change in the mechanical properties and weight. The in vitro hydrolysis was carried out for catguts using collagenase (pH 7.4) and pepsin (pH 1.6), simulating the in vivo environments. The kinetic study on the weight loss of the fibre at the collagenase hydrolysis suggested that the degradation proceeded gradually from the surface of the fibre into the core. The enzymatic hydrolysis was different from the non-enzymatic acidic hydrolysis which resulted in almost homogeneous degradation throughout the cross-section of the fibre from the beginning of the hydrolysis reaction. The rate of weight loss with enzymatic hydrolysis was in good agreement with that predicted under the assumption of continuous erosion from the surface. When the collagen sutures were implanted in the subdermal tissue of rabbits, severe infiltration of macrophages and neutrophils was observed at 4 wk post-implantation, probably because of the degradation products from the implanted sutures. Comparison of the tensile strength decrease with the weight loss observed at the in vivo degradation revealed that enzymatic and non-enzymatic hydrolysis occurred concurrently in the subcutaneous tissue.     

In vitro and in vivo assays on egg white/polyvinyl alcohol/clay nanocomposite hydrogel wound dressings

Novel nanocomposite hydrogel wound dressings on the basis of egg white and polyvinyl alcohol, as matrix, and natural Na-montmorillonite clay, as reinforcing agent, were prepared and their performances on wound healing investigated in vitro and in vivo. In vitro cytotoxicity assay revealed non-cytotoxic activity and excellent biocompatibility level of prepared nanocomposite hydrogel wound dressings. The bacterial penetration assay showed the prepared nanocomposite hydrogel wound dressings are excellent barriers against microorganisms and could protect the wound from infection during the wound healing. In vivo animal study showed that the wound healing process was considerably faster in wounds covered with nanocomposite hydrogel wound dressings compared to the conventional wound dressing, i.e. sterile gauze, due to creation of a moist environment on the wound surface and faster migration rate of the epidermal cells. The mechanical properties of healed wounds with nanocomposite hydrogel wound dressings were better than those control wounds covered with sterile gauze due to their better collagen formation ability as a result of created moist healing condition as well as the presence of egg white, as a source of proteins, in their structures.     

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。     

In vitro and in vivo evaluation of alginate/sol-gel synthesized aminopropyl-silicate/alginate membrane for bioartificial pancreas

Alginate/aminopropyl-silicate/alginate (Alg/AS/Alg) membrane was prepared on Ca-alginate gel beads by a sol-gel process. The membrane has identical to Si-O-Si identical to bonds as well as electrostatic bonds between amino groups of AS and carboxyl groups of alginate. Permeability and stability were investigated for the membrane. Furthermore, rat islets encapsulated in the membrane (499 +/- 32 microns in diameter, 1000 islets/recipient) were transplanted to the peritoneal cavities of the mice with streptozotocin-induced diabetes. Our data show that the membrane had the molecular weight cut-off point of between 70 and 150 kDa, and hardly inhibited the permeation of glucose and insulin. The Alg/AS/Alg microcapsule was more stable than the well-known Alg/poly-L-lysine (PLL)/Alg microcapsule. After 30 days of soaking in stimulated body fluid, the percentages of intact microcapsule were 98.4 +/- 0.5 (mean +/- SEM)% and 88.0 +/- 1.5% (p < 0.001) for the Alg/AS/Alg and Alg/PLL/Alg microcapsules, respectively. The maximum maintenance period of normoglycemia was 105 days without administration of immunosuppressive drugs.