Material design of bioabsorbable inorganic/organic composites for bone regeneration

Two new bioabsorbable inorganic/organic composite materials were developed for bone regeneration. One material used was beta-TCP/PLGC in which poly(L-lactide-co-glycolide-co-epsilon-caprolactone) and beta-tricalcium phosphate were used as the matrix and filler, respectively. The other material used was HAp/Col-a soft nanocomposite of hydroxyapatite and type I collagen. Using these composites, two bone implants were designed. The efficacy of these implants was investigated by applying them to the critical-sized bone defects that were created in the canine tibia. Although no tissue engineering techniques such as application of growth factors or stem cells was utilized, successful healing was observed. These results suggested that bone regeneration in the critical-sized defects is possible without the use of growth factors or stem cells if the materials and the bone implants are suitably designed.     

Gene expressions of Collagen type I,ALP and BMP-4 in osteo-inductive BCP implants show similar pattern to that of natural healing bones

Osteo-inductive materials give rise to ectopic bone formation in vivo either in muscles or in subcutaneous tissue. However, the underlying molecular mechanism is totally unclear. To investigate the expression pattern of bone related genes in osteo-inductive materials, we performed quantitative PCR (qPCR) to detect the expressions of type I collagen, alkaline phosphatase (ALP) and bone morphogenetic protein 4 (BMP-4) in biphasic calcium phosphate (BCP) ceramics implanted in dorsal muscle of dogs. Bone formation in mandibular alveolus defects served as controls showing the expression patterns of these genes in natural healing bones. Histological examinations were performed to show the bone formation in osteo-inductive BCP implants. Data of qPCR indicated that all tested genes had a similar expression pattern with two peaks during the bone formation either in BCP implants or natural healing bones. Type I collagen and ALP were expressed at lower levels with delayed peak in BCP implant than that in natural healing bone. Higher BMP-4 expression level was detected in BCP ceramic implant than in natural healing bone at all the time points. These results demonstrated that expression patterns of bone-related genes in the inductive bone formation are similar to that of natural healing bone formation. As these three genes are important parameters for osteoblast activity in bone formation, our data provide clue to uncover the molecular mechanism of bone formation in osteo-inductive materials.     

双醛羧甲基纤维素-胶原复合止血材料的研制

以羧甲基纤维素钠(CMC)和胶原(Col)为原料,通过选择性氧化制得双醛羧甲基纤维素钠(DCMC),并对胶原进行交联改性,制得一种新型胶原止血海绵DCMC-Col。运用红外光谱、圆二色谱、差示扫描量热仪、原子力显微镜、扫描电镜对改性后的DCMC-Col复合海绵进行检测。结果表明,改性后胶原的热稳定性得到明显提升,且胶原三股螺旋结构不会遭到破坏。全凝血时间检测表明,当DCMC用量为15%时,海绵的止血性能最优。吸水率、溶血率和MTT实验结果表明,DCMC-Col复合海绵的各项指标均满足止血材料的要求,且细胞相容性良好。因此,DCMC-Col复合海绵是一种具有潜在医学应用价值的新型止血材料。     

Fabrication of pre-determined shape of bone segment with collagen-hydroxyapatite scaffold and autogenous platelet-rich plasma

Background Reconstruction of large segment of bony defects is frequently needed in hand surgery. Autogenous bone grafting is considered the standard in management of these bony defects but has limited source of graft material. Collagen and hydroxyapatite have been used as bone-filling materials and are known to serve as the osteoconductive scaffold for bone regeneration. On the other hand, platelet-rich plasma is a kind of natural source of growth factors, and has been used successfully in bone regeneration and improving wound healing. This study was designed to evaluate the effectiveness of using biohybrids of platelet-rich plasma and collagen-hydroxyapatite beads for fabricating of protrusive bone in a rabbit animal model. Methods Biomaterial beads comprised of particulate hydroxyapatite dispersed in fibrous collagen (type I) matrices were prepared and filled in the ringed polytetrafluoroethylene (PTFE) artificial vascular graft (3 cm long, 1 cm in diameter). New Zealand White rabbits were each implanted with two cylindrical PTFE artificial vascular graft over both iliac crests (n = 16). A 2 × 0.5 cm opening was created at the side of each PTFE chamber to allow the content of chamber in contact with the bone marrow of the ileum. The chambers were empty (groups A and D), filled with type I collagen/hydroxyapatite beads (groups B and C). In groups C and D, autologous platelet rich plasma (PRP) was given by transcutaneous injection method into the chambers every week. After 12 weeks, the animals were sacrificed and the chambers were harvested for radiological and histological analysis. Results In plain radiographs, the group C chambers had significantly higher bone tissue engineered (average calcified density 0.95, average calcified area 61.83%) compared with other groups (P < 0.001). In histological examination, there was a creeping substitution of the implant by the in-growth of fibrovascular tissue in group C. Abundant fibrovascular networks positioned interstitially between these biomaterial beads in all parts of chamber. Degradation of these beads and newly formed capillaries and osteoids around the degraded matrixes are shown. The continually calcification in the matrixes or degraded matrixes is evidenced by the strong green fluorescence observed under the confocal microscope. In group B, looser architecture without evidence of tissue in-growth was shown. In the scaffold absent groups (A and D), there was only fibrous tissue shown within the chamber. Conclusions In this study, we have demonstrated a feasible approach to fabricate an osseous tissue that meets clinical need. Using the type I collagen/ hydroxyapatite gel beads matrixes and intermittent injection of autologous platelet-rich-plasma, specific 3D osseous tissues with fibrovascular network structure from pre-exist bony margin were successfully created in an in vivo animal model.     

Developments in injectable multiphasic biomaterials. The performance of microporous biphasic calcium phosphate granules and hydrogels

Calcium phosphate bioceramic granules associated with hydrosoluble polymers were developed as bone substitutes for various maxillofacial and orthopaedic applications. These injectable bone substitutes, support and regenerate bone tissue and resorb after implantation. The efficiency of these multiphasic materials is due to the osteogenic and osteoconductive properties of the microporous biphasic calcium phosphate. The associated hydrosoluble polymers are considered as carriers in order to achieve the rheological properties of injectable bone substitutes (IBS). In this study, we used 2 semi synthetic hydrosoluble polymers of polysaccharidic origin. The hydroxy propyl methyl cellulose (HPMC), with and without silane, was combined with microporous BCP granules. The presence of silane induced considerable gelation of the suspension. The 2 IBS used (without gelation, IBS1, with gelation, IBS2) were implanted in critical size femoral epiphysis defects in rabbits. No foreign body reactions were observed in either sample. However, because of the higher density from gelation, cell colonisation followed by bone tissue ingrowth was delayed over time with IBS2 compared to the IBS1 without gelation. The results showed resorption of the BCP granule and bone ingrowth at the expense of both IBS with different kinetics. This study demonstrates that the hydrogel cannot be considered merely as a carrier. The gelation process delayed cell and tissue colonisation by slow degradation of the HPMC Si, compared to the faster release of HPMC with IBS1, in turn inducing faster permeability and spaces for tissue ingrowth between the BCP granules.     

Osteopenic bone cell response to strontium-substituted hydroxyapatite

Ionic substitution is a powerful tool to improve the biological performance of calcium phosphate based materials. In this work, we investigated the response of primary cultures of rat osteoblasts derived from osteopenic (O-OB) bone to strontium substituted hydroxyapatite (SrHA), and to hydroxyapatite (HA) as reference material, compared to normal (N-OB) bone cells. Strontium (Sr) and calcium (Ca) cumulative releases in physiological solution are in agreement with the greater solubility of SrHA than HA, whereas the differences between the two materials are levelled off in DMEM, which significantly reduced ion release. O-OB cells grown on SrHA exhibited higher proliferation and increased values of the differentiation parameters. In particular, Sr substitution increased the levels of proliferation, alkaline phosphatase, and collagen type I, and down-regulated the production of interleukin-6 of O-OB cells, demonstrating a promising future of SrHA in the treatment of bone lesions and defects in the presence of osteoporotic bone.     

Nano-hydroxyapatite—Cellulose acetate composites for growing of bone cells

Natural bone consists of natural polymers, collagen fibers and nanocrystals of minerals, mainly nano-hydroxyapatite (n-HA). Bone cells, which maintain the activities and metabolism of bone, are supported by and interact with this organic-inorganic hybrid matrix in nature. Artificial bone tissue scaffolds based on natural hybrids of cellulose acetate (CA) and nano-hydroxyapatite (n-HA) were fabricated in a bio-mimicking 3D matrix architecture using a single step nanomanufacturing technique and were used for in-vitro bone regeneration studies for up to 14 days. Osteoblasts grown on these scaffolds were found to interact strongly with the HA nanoclusters that were uniformly distributed on the CA fibers, promoting cell elongation, growth and phenotype retention. Hexagonal apatite crystals were shown to crystallize on the n-HA seeds. The natural, open, hybrid, 3D nanoscaffolds thus appear to be most promising bone repair agents.     

The mechanical properties of calcium phospate ceramics modified by collagen coating and populated by osteoblasts

Bioceramics containing hydroxyapatite (HA), tricalcium phosphate (TCP) or composites which combine the best properties of both materials are among the principal candidates for bone replacement grafts. In this study we have investigated the mechanical strength of HA, TCP and composites of the two in the ratios 75;25 (H75), 50:50 (H50) and 25:75 (H25). The strength of each material was investigated in the presence and absence of collagen coating, and the influence of osteoblast culture for up to 28 days on strength was determined. TCP, H25 and H75 were significantly weakened by collagen coating, the strengths of the other materials were either not affected (HA) or increased (H50). Culture with osteoblasts significantly increased the strength of uncoated HA and H50, but this effect was not observed when the materials were coated with collagen. Our results indicate that ceramic composition affects the interactions between collagen coating, culture with osteoblasts and mechanical strength of the material. Although collagen coating has been found to increase the proliferation of osteoblasts into these ceramic materials, it may be necessary to stabilise and optimise the coating process to minimise effects on mechanical strength.     

Microstructural evolution in external callus of human long bone

Accurate knowledge of bone fracture healing process is of clinical and theoretical importance in bone repair and regeneration, and biomineralization. It is well known that the histological healing occurs via the formation of hematoma, fibrocartilage, bony callus and bone modeling/remodeling. However, the detailed process from fracture to healing at the microstructural level remains unclear. In the present study, an evolutionary model of external callus is proposed, in which five representative stages are presented in terms of the organization of collagen and minerals during the formation of bony callus. The first stage is the formation of loose, disordered collagen fibrils, which is followed by mineralization on some of these individual microfibrils. Then the matrix is characterized by the fusion of mineralized individual fibrils into bundles. In the third stage, the absorption of disordered matrix occurs. This is gradually replaced by ordered collagen in stage four. Finally, completely ordered mineralized tissue is formed. The proper sequence of the process plays an important role in deciding the success of healing.In addition to the common mineral phase of hydroxyapatite (HA), dicalcium phosphate dihydrate (DCPD) phase was also found in early stage of healing, especially in rapid healing (children's callus). It vanished in the following process of healing. The deposition of DCPD is supposed to be brought about by some non-collagenous protein.     

Preparation of a biphasic scaffold for osteochondral tissue engineering

Tissue engineering has been developed as a prospective approach for the repair of articular cartilage defects. Engineered osteochondral implants can facilitate the fixation and integration with host tissue, and therefore promote the regeneration of osteochondral defects. A biphasic scaffold with a stratified two-layer structure for osteochondral tissue engineering was developed from biodegradable synthetic and naturally derived polymers. The upper layer of the scaffold for cartilage engineering was collagen sponge; the lower layer for bone engineering was a composite sponge of poly(DL-lactic-co-glycolic acid) (PLGA) and naturally derived collagen. The PLGA–collagen composite sponge layer had a composite structure with collagen microsponge formed in the pores of a skeleton PLGA sponge. The collagen sponge in the two respective layers was connected. Observation of the collagen/PLGA–collagen biphasic scaffold by scanning electron microscopy (SEM) demonstrated the connected stratified structure. The biphasic scaffold was used for culture of canine bone-marrow-derived mesenchymal stem cells. The cell/scaffold construct was implanted in an osteochondral defect in the knee of a one-year old beagle. Osteochondral tissue was regenerated four months after implantation. Cartilage- and bone-like tissues were formed in the respective layers. The collagen/PLGA–collagen biphasic scaffold will be useful for osteochondral tissue engineering.     

分级多孔壳聚糖/聚乳酸复合支架的制备及骨修复性能

为了仿生莲藕内部的贯穿大孔结构,以生物相容性好的壳聚糖(CS)作为基质材料,利用冰粒致孔、石蜡模具和冰模具成型3种成型方法制备了分级多孔CS支架材料,然后与力学强度较高的聚乳酸(PLLA)复合,制备网络互穿CS/PLLA复合支架。通过SEM、压缩强度测试和兔股骨髁骨缺损模型对CS/PLLA复合材料的形貌、力学强度和骨修复性能进行了表征。结果表明:利用冰模具制备的CS/PLLA复合支架能可控、批量制备,具有微米-毫米分级多孔结构,大孔孔径约为2mm,内部均匀分布着孔径约为60μm的贯穿微孔,并在微孔内形成密集的PLLA絮状网络结构。干态复合材料的压缩强度和模量分别比纯CS支架的提高了6倍和15倍。体内植入实验结果表明,CS/PLLA复合材料能够促进骨缺损的愈合,并随着新骨的形成,复合材料逐渐被降解吸收。     

Evaluation of Injectable Composite Material Comprising Biphasic Bone Substitutes and Crosslinked Collagen

Bone tissue engineering has emerged as a promising approach to regenerate bone tissue, and injectable biomaterials have shown potential for bone regeneration applications due to their ease of administration and ability to fill irregularly shaped defects. This study aims to develop and characterize an injectable composite material comprising biphasic bone substitutes (BBS) and crosslinked porcine collagen type I for bone regeneration applications. The collagen is crosslinked via a UVA-riboflavin crosslinking strategy and evaluated by testing the physicochemical properties, including the rheological behavior, dynamic storage modulus (G′) and loss modulus (G″), and in vitro degradation process. The results show that the crosslinked collagen (xCol) exhibits suitable physicochemical properties for injectability and improved viscoelasticity and degradation resistance. Furthermore, xCol is then combined with BBS in a predetermined ratio, obtaining the injectable composite material. The biocompatibility of the materials is evaluated in vitro by XTT and BrdU assays on fibroblasts and preosteoblasts. The results demonstrate that the composite material is biocompatible and supporting pre-osteoblasts proliferation. In conclusion, the injectable composite material BBS-xCol has promising physiochemical and biological properties for bone regeneration applications. Further studies are warranted to evaluate its efficacy in vivo and optimize its composition for clinical translation.     

Carbon nanotubes for orthopaedic implants

The physical and biological limitations of current orthopaedic implant materials are a major challenge for bone tissue engineering. Nanotechnology has introduced new materials and methods for meeting this challenge. The application of nanotechnology to engineering new bone substitutes finds a model in the nanoscale components of natural bone tissue. Carbon nanotubes are a macromolecular form of carbon with exceptional properties and similar morphology and dimensions to the nanoscale collagen fibers of natural bone tissue. Carbon nanotubes have been used in two main areas of bone tissue engineering: for structural and electrical enhancement of polymer and ceramic composites and for nanostructured coatings to improve the bioactivity of implant surfaces. By incorporating carbon nanotubes into the design and engineering of bone tissue substitutes, researchers have attempted to overcome limitations in the structural and biological compatibility of traditional orthopaedic implant materials.     

Modelling the mechanics of partially mineralized collagen fibrils,fibres and tissue

Progressive stiffening of collagen tissue by bioapatite mineral is important physiologically, but the details of this stiffening are uncertain. Unresolved questions about the details of the accommodation of bioapatite within and upon collagen''s hierarchical structure have posed a central hurdle, but recent microscopy data resolve several major questions. These data suggest how collagen accommodates bioapatite at the lowest relevant hierarchical level (collagen fibrils), and suggest several possibilities for the progressive accommodation of bioapatite at higher hierarchical length scales (fibres and tissue). We developed approximations for the stiffening of collagen across spatial hierarchies based upon these data, and connected models across hierarchies levels to estimate mineralization-dependent tissue-level mechanics. In the five possible sequences of mineralization studied, percolation of the bioapatite phase proved to be an important determinant of the degree of stiffening by bioapatite. The models were applied to study one important instance of partially mineralized tissue, which occurs at the attachment of tendon to bone. All sequences of mineralization considered reproduced experimental observations of a region of tissue between tendon and bone that is more compliant than either tendon or bone, but the size and nature of this region depended strongly upon the sequence of mineralization. These models and observations have implications for engineered tissue scaffolds at the attachment of tendon to bone, bone development and graded biomimetic attachment of dissimilar hierarchical materials in general.     

Mechanical performance and architecture of biocomposite honeycombs and foams from core–shell holocellulose nanofibers

CNFs (cellulose nanofibers) based on holocellulose have a pure cellulose fibril core, with a hemicellulose coating. The diameter is only around 6–8 nm and the hemicellulose surface coating has anionic charge. These CNFs are used to prepare honeycomb and foam structures by freeze-drying from dilute hydrocolloidal suspensions. The materials are compared with materials based on “conventional” cellulose CNFs from sulfite pulp with respect to mechanical properties in compression. Characterization methods include FE-SEM of cellular structure, and the analysis includes comparisons with similar materials from other types of CNFs and data in the literature. The honeycomb structures show superior out-of-plane properties compared with the more isotropic foam structures, as expected. Honeycombs based on holocellulose CNFs showed better properties than sulfite pulp CNF honeycombs, since the cellular structure contained less defects. This is related to better stability of holocellulose CNFs in colloidal suspension.     

Self-assembly of mineralized collagen composites

This paper presents a review of the current understanding of the structure, self-assembly mechanisms, and properties of mineralized collagen fibril composites in connective tissues, such as in lamellar bones, woven bones, zebrafish skeletal bone, and ivory. Recent work involving biomimetic synthesis of new materials with the structure of mineralized collagen is described. The focus in the paper is mainly on materials containing type I collagen, with mineralization by Ca–P crystals although some other systems are also described. Investigation and simulation of naturally occurring fibril structures can offer some new ideas in the design and fabrication of new functional materials, for applications such as bone grafts or for use as scaffolds in tissue engineering and biomimetic engineering materials. The development of bone grafts based on the mineralization of self-assembled collagen fibrils in vivo and in vitro is an active area of research. This kind of bone graft composite has already shown great promise and success in clinical applications, on account of its compositional and structural similarity to autologous bone. It is suggested that future work in this should focus on both basic theoretical aspects as well as the development of applications. In particular issues including control of morphology, incorporation of foreign ions, interaction with biomolecules, and the assembly of organic and inorganic phases are all still not well understood. In the area of applications, the design of composite materials with a hierarchical structure closer to that of natural hard tissues, and the synthesis of bone grafts and tooth regenerative materials, as well as biomimetic functional materials, are areas currently being examined by many research groups.     

骨组织工程支架材料的研究进展

综述了骨组织工程支架材料在骨缺损治疗的研究现状,并展望了其未来的发展方向。利用各种材料的优势互补性,将两种或两种以上材料通过恰当的方式组合成复合支架材料,其力学性能和降解速率可根据各组分材料的种类、数量及组合方法的变化进行调节,按照要求制备出具有一定机械强度和降解速率的支架材料。并指出骨组织工程复合支架材料将会成为骨缺损修复中一类富有潜力的生物材料。     

Treatment of periodontal defects in dogs using an injectable composite hydrogel/biphasic calcium phosphate

An injectable composite silanized hydroxypropyl methyl cellulose/biphasic calcium phosphate (Si-HPMC/BCP) has been investigated in humans with promising results. The aim of this study was to evaluate his efficacy for treating periodontal defects (canine fenestration and premolar furcation) in dog models. At 3?months, we observed that bone formation around BCP particles in furcation model is more discernible but not statistically significant in defects filled with Si-HPMC/BCP compared to healing in control. We suggest that BCP particles sustain the bone healing process by osteoconduction, while the Si-HPMC hydrogel enhances intergranular cohesion and acts as an exclusion barrier. Furthermore, bone ingrowth is not so distinctive in superficial defects where the biomaterial appears unstable. These results with Si-HPMC/BCP are encouraging. In addition, this biomaterial is easy to use and simplifies the process of filling periodontal lesions. However, more researches are needed to improve the viscosity and hardness to adjust the material to the specificities of periodontal defects.     

Theoretical and computational hierarchical nanomechanics of protein materials: Deformation and fracture

Proteins constitute the building blocks of biological materials such as tendon, bone, skin, spider silk or cells. An important trait of these materials is that they display highly characteristic hierarchical structures, across multiple scales, from nano to macro. Protein materials are intriguing examples of materials that balance multiple tasks, representing some of the most sustainable material solutions that integrate structure and function. Here we review progress in understanding the deformation and fracture mechanisms of hierarchical protein materials by using a materials science approach to develop structure-process-property relations, an effort defined as materiomics. Deformation processes begin with an erratic motion of individual atoms around flaws or defects that quickly evolve into formation of macroscopic fractures as chemical bonds rupture rapidly, eventually compromising the integrity of the structure or the biological system leading to failure. The combination of large-scale atomistic simulation, multi-scale modeling methods, theoretical analyses combined with experimental validation provides a powerful approach in studying deformation and failure phenomena in protein materials. Here we review studies focused on the molecular origin of deformation and fracture processes of three types of protein materials. The review includes studies of collagen - Nature’s super-glue; beta-sheet rich protein structures as found in spider silk - a natural fiber that can reach the strength of a steel cable; as well as intermediate filaments - a class of alpha-helix based structural proteins responsible for the mechanical integrity of eukaryotic cells. The article concludes with a discussion of the significance of universally found structural patterns such as the staggered collagen fibril architecture or the alpha-helical protein motif.     

Preparation of flexible bone tissue scaffold utilizing sea urchin test and collagen

Gonads of sea urchin are consumed in Japan and some countries as food and most parts including its tests are discarded as marine wastes. Therefore, utilization of them as functional materials would reduce the waste as well as encourage Japanese fishery. In this study, magnesium containing calcite granules collected from sea urchin tests were hydrothermally phosphatized and the obtained granules were identified as approximately 82% in mass of magnesium containing β-tricalcium phosphate and 18% in mass of nonstoichiometric hydroxyapatite, i.e., a biphasic calcium phosphate, maintaining the original porous network. Shape-controlled scaffolds were fabricated with the obtained biphasic calcium phosphate granules and collagen. The scaffolds showed good open porosity (83.84%) and adequate mechanical properties for handling during cell culture and subsequent operations. The MG-63 cells showed higher proliferation and osteogenic differentiation in comparison to a control material, the collagen sponge with the same size. Furthermore, cell viability assay proved that the scaffolds were not cytotoxic. These results suggest that scaffold prepared using sea urchin test derived calcium phosphate and collagen could be a potential candidate of bone void fillers for non-load bearing defects in bone reconstruction as well as scaffolds for bone tissue engineering.