骨髓基质细胞-成骨细胞复合培养体系的建立

背景:在骨修复和重建过程中,成骨细胞和骨髓基质细胞是主要功能细胞,二者存在着密切的功能联系。目的:通过骨髓基质细胞-成骨细胞共育体系的建立,观察共育体系中两种细胞之间的功能影响及生物学特点。方法:原代分离人骨髓基质细胞和人成骨细胞,将2种细胞置于Transwell共育环境中共同培养,建立人骨髓基质细胞-成骨细胞共育体系。分别采用MTT、丫啶橙染色、碱性磷酸酶活性检测等方法初步评价共育体系中两种细胞增殖、凋亡及功能改变情况。结果与结论:在复合培养体系中,骨髓基质细胞-成骨细胞共育体系能够促进成骨细胞的增殖与碱性磷酸酶的活性,同时抑制骨髓基质细胞的凋亡,促进骨髓基质细胞的趋化聚集。结果提示在复合培养体系中,骨髓基质细胞能够加速成骨细胞的增殖及成骨活性,另外成骨细胞也可减少骨髓基质细胞的凋亡,并加强其成骨性分化的作用。两者之间具有较为紧密的影响和功能联系。

中国生物材料学会骨修复材料与器械分会2014年全国骨科材料与器械产学研医管交流会通知

依据《国家中长期科学与技术发展规划纲要》的方针政策,针对我国当前生物医用材料及医疗器械行业的发展现状,着眼于我国骨科植物材料及器械发展的长远规划,中国生物材料学会骨修复材料与器械分会将召开'2014年全国骨科材料与器械产学研医管交流会'。本次交流会立足临床实际需求,融合产-学-研-医-管各产业链优势资源,通过各专题会形式充分研讨骨材料与器械国内外前沿科学技术,提炼临床急需解决的共性关键器械问题及解决方案,为我国十三五相关规划的制订建言献策。

Recent Progress in Cartilage Tissue Engineering--Our Experience and Future Directions

Given the limited spontaneous repair that follows cartilage injury, demand is growing for tissue engi- neering approaches for cartilage regeneration. There are two major applications for tissue-engineered cartilage. One is in orthopedic surgery, in which the engineered cartilage is usually used to repair cartilage defects or loss in an articular joint or meniscus in order to restore the joint function. The other is for head and neck reconstruction, in which the engineered cartilage is usually applied to repair cartilage defects or loss in an auricle, trachea, nose, larynx, or eyelid. The challenges faced by the engineered car- tilage for one application are quite different from those faced by the engineered cartilage for the other application. As a result, the emphases of the engineering strategies to generate cartilage are usually quite different for each application. The statuses of preclinical animal investigations and of the clinical translation of engineered cartilage are also at different levels for each application. The aim of this review is to provide an opinion piece on the challenges, current developments, and future directions for cartilage engineering for both applications.

Osteogenic potential of human periosteum-derived progenitor cells in PLGA scaffold using allogeneic serum

The use of periosteum-derived progenitor cells （PCs） combined with bioresorbable materials is an attractive approach for tissue engineering. The aim of this study was to characterize the osteogenic differentiation of PC in 3-dimensional （3D） poly-lactic-co-glycolic acid （PLGA） fleeces cultured in medium containing allogeneic human serum. PCs were isolated and expanded in monolayer culture. Expanded cells of passage 3 were seeded into PLGA constructs and cultured in osteogenic medium for a maximum period of 28 d. Morphological, histological and cell viability analyses of three-dimensionally cultured PCs were performed to elucidate osseous synthesis and deposition of a calcified matrix. Furthermore, the mRNA expression of type Ⅰ collagen, osteocalcin and osteonectin was semi-quantitively evaluated by real-time reverse transcriptase-polymerase chain reaction （RT-PCR）. The fibrin gel immobilization technique provided homogeneous PCs distribution in 3D PLGA constructs. Live-dead staining indicated a high viability rate of PCs inside the PLGA scaffolds. Secreted nodules ofneo-bone tissue formation and the presence of matrix mineralization were confirmed by positive yon Kossa staining. The osteogenic differentiation of PCs was further demonstrated by the detection of type I collagen, osteocalcin and osteonectin gene expression. The results of this study support the concept that this tissue engineering method presents a promising method for creation of new bone in vivo.

Tethering of Gly-Arg-Gly-Asp-Ser-Pro-Lys Peptides on Mg-Doped Hydroxyapatite

Stem cell homing, namely the recruitment of mesenchymal stem cells （MSCs） to injured tissues, is highly effective for bone regeneration in vivo. In order to explore whether the incorporation of mimetic peptide sequences on magnesium-doped （Mg-doped） hydroxyapatite （HA） may regulate the homing of MSCs, and thus induce cell migration to a specific site, we covalently functionalized MgHA disks with two chemotactic/haptotactic factors： either the fibronectin fragment III1-C human （FF III1-C）, or the peptide sequence Gly-Arg-Gly-Asp-Ser-Pro-Lys, a fibronectin analog that is able to bind to integrin trans- membrane receptors. Preliminary biological evaluation of MSC viability, analyzed by 3-（4,5-dimethyl- thiazol-2-yl）-2,5-diphenyltetrazolium bromide （MTT） test, suggested that stem cells migrate to the MgHA disks in resoonse to the grafted haototaxis stimuli.

Preparation of new tissue engineering bone-CPC/PLGA composite and its application in animal bone defects

To investigate the feasibility of implanting the biocomposite of calcium phosphate cement(CPC)/polylactic acid-polyglycolic acid(PLGA) into animals for bone defects repairing,the biocomposite of CPC/PLGA was prepared and its setting time,compressive strength,elastic modulus,pH values,phase composition of the samples,degradability and biocompatibility in vitro were tested.The above-mentioned composite implanted with bone marrow stromal cells was used to repair defects of the radius in rabbits.Osteogenesis was histomorphologically observed by using an electron-microscope.The results show that compared with the CPC,the physical and chemical properties of CPC/PLGA composite have some differences in which CPC/PLGA composite has better biological properties.The CPC/PLGA composite combined with seed cells is superior to the control in terms of the amount of new bones formed after CPC/PLGA composite is implanted into the rabbits,as well as the speed of repairing bone defects.The results suggest that the constructed CPC/PLGA composite basically meets the requirements of tissue engineering scaffold materials and that the CPC/PLGA composite implanted with bone marrow stromal cells may be a new artificial bone material for repairing bone defects because it can promote the growth of bone tissues.

Study on biocompatibility of PDLLA/HA/DBM with co-cultured human osteoblasts in vitro

Objective: To evaluate the osteocompatibility of D, L-polylactic/hydroxyapatite/decalcifying bone matrix (PDLLA/HA/DBM), and compare with PDLLA and DBM. Methods: Human primary osteoblasts isolated from the femoral head of patients were inoculated onto PDLLA/HA/DBM, PLA and DBM respectively. The proliferation rate and collagen Ⅰ expression were detected. The interface between biomaterial and osteoblasts was investigated with phase contrast microscopy and electron scanning microscopy. Results: Best proliferation rate was observed with the PDLLA/HA/DBM and followed by DBM and PLA, suggesting that PDLLA/HA/DBM satisfying most requirements for the cultivation of human osteoblasts. Scanning electron microscopy showed the morphology of osteoblasts was correlated with the proliferation data. The cells, well spread and flattened, were attached closely on the surface of biomaterial with an arched structure and had normal morphology. The extracellular collagenous matrixs covered the surface of biomaterial and packed the granules of biomaterial. Conclusion: PDLLA/HA/DBM can form osteointerface early and have a good biocompability.

Influence of different gravitational environments on the mechanotrans-duction in osteoblasts

Objective: To observe the effects of different gravitational environments on release of prostaglandin E2 in rat calvarial osteoblasts induced by fluid shear stress (FSS) so that to investigate the influence of different gravity on mechanotransduction in osteoblasts. Methods: Osteoblasts were isolated from neonatal rat calvariae and then were set to three groups. Each was cultured in one gravitational environment; 1G terrestrial gravitational environment (control), simulated weightlessness achieved by using clinostat and 3G gravitational environment achieved by using centrifuge for 60 h, then osteoblasts were treated with 0. 5 Pa or 1. 5 Pa FSS in a flow chamber for 1 h. The release of PGE2 in osteoblasts was determined. Results: In 1G gravitational environment, the release of PGE2 was significantly increased along with the sustaining of FSS treatments (P<0. 01), but there was no remarkable difference between the responses to 0. 5 Pa FSS and 1. 5 Pa FSS (P>0. 05). While in simulated weightlessness environment group, no detectable release of PGE2 was found with the treatment of 0. 5 Pa FSS (P<0. 01), and the release of PGE2 was delayed and the amount of PGE2 production was remarkably decreased with 1. 5 Pa FSS treatment as compared with that of 1G group (P<0. 01). The responsiveness of osteoblasts cultured in 3G gravitational environment to FSS was similar to that of 1G group. Conclusion: These results indicate that in vitro the mechanotransduction in osteoblasts iss affected by stimulated weightlessness, whereas it is not altered in 3G gravitational environment.

Microstructure and Mechanical Properties of Calcium Phosphate Cement/gelatine Composite Scaffold with Oriented Pore Structure for Bone Tissue Engineering

In this study,the macroporous calcium phosphate cement with oriented pore structure was prepared by freeze casting.SEM observation showed that the macropores in the porous calcium phosphate cement were interconnected aligned along the ice growth direction.The porosity of the as-prepared porous CPC was measured to be 87.6% by Archimede's principle.XRD patterns of specimens showed that poorly crystallized hydroxyapatite was the main phase present in the hydrated porous calcium phosphate cement.To improve the mechanical properties of the CPC scaffold,the 15% gelatine solution was infiltrated into the pores under vacuum and then the samples were freeze dried to form the CPC/gelatine composite scaffolds.After reinforced with gelatine,the compressive strength of CPC/gelatin composite increased to 5.12 MPa,around 50 times greater than that of the unreinforced macroporous CPC scaffold,which was only 0.1 MPa.And the toughness of the scaffold has been greatly improved via the gelatine reinforcement with a much greater fracture strain.SEM examination of the specimens indicated good bonding between the cement and gelatine.In conclusion,the calcium phosphate cement/gelatine composite with oriented pore structure prepared in this study might be a potential scaffold for bone tissue engineering.

TISSUE-ENGINEERED GRAFT CONSTRUCTED BY SELF-DERIVED BONE MARROW MONONUCLEAR CELLS AND HETEROGENEOUS ACELLULARIZED TISSUE MATRIX

Objective To create a method for constructing a tissue-engineered graft with self-derived bone marrow cells and heterogeneous acellular matrix.Methods The mononuclear cells were isolated from bone marrows drawn from piglets and cultured in different mediums including either vascular endothelial growth factor(VEGF)or platelet derived growth factor BB(PDGF-BB)to observe their expansion and differentiation.The aortas harvested from canines were processed by a multi-step decellularizing technique to erase.The bone marrow mononuclear cells cultured in the mediums without any growth factors were seeded to the acellular matrix.The cells-seeded grafts were incubated in vitro for 6 d and then implanted to the cells-donated piglets to substitute parts of their native pulmonary arteries.Results After 4 d culturing,the cells incubated in the medium including VEGF showed morphological feature of endothelial cells(ECs)and were positive to ECs-specific monoclonal antibodies of CD31,FLK-1,VE-Cadherin and vWF.The cells incubated in the medium including PDGF-BB showed morphological feature of smooth muscle cells(SMCs)and were positive to SMCs-specific monoclonal antibodies of α-SMA and Calponin.One hundred days after implantation of seeded grafts,the inner surfaces of explants were smooth without thrombosis,calcification and aneurysm.Under the microscopy,plenty of growing cells could be seen and elastic and collagen fibers were abundant.Conclusion Mesenchymal stem cells might exist in mononuclear cells isolated from bone marrow.They would differentiate into endothelial cells or smooth muscle cells in proper in vitro or in vivo environments.The bone marrow mononuclear cells might be a choice of seeding cells in constructing tissue-engineered graft.

Repair of articular cartilage defects in rabbits through tissue-engineered cartilage constructed with chitosan hydrogel and chondrocytes

Objective： In our previous work, we prepared a type of chitosan hydrogel with excellent biocompatibility. In this study, tissue-engineered cartilage constructed with this chitosan hydrogel and costal chondrocytes was used to repair the articular cartilage defects. Methods： Chitosan hydrogels were prepared with a crosslinker formed by combining 1,6-diisocyanatohexane and polyethylene glycol. Chitosan hydrogel scaffold was seeded with rabbit chondrocytes that had been cultured for one week in vitro to form the preliminary tissue-engineered cartilage. This preliminary tissue-engineered cartilage was then transplanted into the defective rabbit articular cartilage. There were three treatment groups： the experimental group received preliminary tissue-engineered cartilage; the blank group received pure chitosan hydrogels; and, the control group had received no implantation. The knee joints were harvested at predetermined time. The repaired cartilage was analyzed through gross morphology, histologically and immunohistochemically. The repairs were scored according to the international cartilage repair society （ICRS） standard. Results： The gross morphology results suggested that the defects were repaired completely in the experimental group after twelve weeks. The regenerated tissue connected closely with subchondral bone and the boundary with normal tissue was fuzzy. The cartilage lacuna in the regenerated tissue was similar to normal cartilage lacuna. The results of ICRS gross and histological grading showed that there were significant differences among the three groups （P〈0.05）. Conclusions： Chondrocytes implanted in the scaffold can adhere, proliferate, and secrete extracellular matrix. The novel tissue-engineered cartilage constructed in our research can completely repair the structure of damaged articular cartilage.

Rapid prototyping technology and its application in bone tissue engineering

Bone defects arising from a variety of reasons cannot be treated effectively without bone tissue reconstruction.Autografts and allografts have been used in clinical application for some time,but they have disadvantages.With the inherent drawback in the precision and reproducibility of conventional scaffold fabrication techniques,the results of bone surgery may not be ideal.This is despite the introduction of bone tissue engineering which provides a powerful approach for bone repair.Rapid prototyping technologies have emerged as an alternative and have been widely used in bone tissue engineering,enhancing bone tissue regeneration in terms of mechanical strength,pore geometry,and bioactive factors,and overcoming some of the disadvantages of conventional technologies.This review focuses on the basic principles and characteristics of various fabrication technologies,such as stereolithography,selective laser sintering,and fused deposition modeling,and reviews the application of rapid prototyping techniques to scaffolds for bone tissue engineering.In the near future,the use of scaffolds for bone tissue engineering prepared by rapid prototyping technology might be an effective therapeutic strategy for bone defects.

Preparation and physicochemical properties of scaffold materials of heterogeneous deproteinized bone

Objective ： To prepare and observe the physicochemical properties of scaffold materials of heterogeneous deproteinized tissue-engineered bone. Methods： Deproteinized bone was made through a series of physicochemical treatments in pig ribs and analyzed with histological observation, scanning electron microscopy, infrared spectrum, X-ray diffraction and energy dispersive analysis, Kjeidahl determination and mechanics analysis. Results： Interstitial collagen fiber was positive and mucin was negative in deproteinized bone, but, both were positive in fresh bone. Deproteinized bone maintained natural pore network. Its pore size was 472.51μm ± 7.02μm and the porosity was 78.15 % ± 6.45 %. The results of infrared spectrum showed that collagen was present in deproteinized bone. Both fresh and deproteinized bone had curve of hydroxyapatite. The Ca/P ratios were 1.71 ± 0. 95 and 1.68 ± 0. 76 （ P 〉 0. 05 ）, and the protein contents were26.6% ± 2.23% and 19.1% ± 2.14% （P 〈 0.05） in fresh and deproteinized bone, respectively. There was no significant difference of destruction load under compression and maximal destruction load between fresh and deproteinized bone （P 〉 0. 05）. The elastic modulus was higher in deproteinized bone than that in fresh bone （P 〈 0.05）. Conclusions ： Physicochemical properties and mechanic strength of deproteiulzed tissue-engineered bone meet the demands of ideal scaffold materials. But, its immunogenicity should be observed through further experiments for its clinical applications.

Reconstruction of caprine mandibular segmental defect by tissue engineered bone reinforced by titanium reticulum

Objective ：To investigate the feasibility of using natural poritos as scaffolds in bone tissue engineering （TE） and repair of caprine mandibular segmental defect with titanium reticulum reinforced. Methods： Natural poritos with a pore of 190-230 μm in size and porosity of about 50 %-65 % was molded into the shape of granules 5 mm × 5 mm × 5 mm in size. Expanded autologous caprine marrow mesenchymal stem cells were induced by recombinant human morphogenetic protein-2 （rhBMP2） to improve osteoblastic phenotype. Then marrow derived osteoblasts were seeded into poritos in density of 4 × 10^7/ml and incubated in vitro for 48 hours prior to implantation. Then osteoblastic cells/poritos complexes were implanted into mandibular defect and the defect was reinforced by titanium reticulum. Implantation of poritos alone acted as the control. Bone regeneration was assessed 4, 8, 16 weeks after implantation using roentgenographie analysis and histological observation was done after 16 weeks. Results： New bone could be observed histologically on the surface and in the pores of natural coral in all specimens in the cell-seeding group, whereas in the control group there was no evidence of osteogenesis process in the center of the construction. The results showed that new bone grafts were successfully restored 16 weeks after implantation. Conclusions： This study suggests the feasibility of using porous coral as scaffold material transplanted with marrow derived osteoblasts by TE method. By means of titanium reticulum reinforcement, mandibular defect could be successfully restored. It shows the potentiality of using this method for the reconstruction of bone defect in cfinic.

Cytocompatible 3D chitosan/hydroxyapatite composites endowed with antibacterial properties:toward a self-sterilized bone tissue engineering scaffold

Bone tissue scaffolds based on bioactive polymer–hydroxyapatite composites have caused infections that seriously limit their extended application. In this study, we proposed a practical ion substitution method to synthesize in situ silver phosphate on the surface of a two-level, threedimensional chitosan/nano-hydroxyapatite scaffold. A release test of silver ions in a phosphate buffered saline(PBS) solution was performed to demonstrate that silver ions were released continuously from the silver phosphate during the initial 6 days of the study. The antibacterial property and cytocompatibility of the scaffolds treated with different concentrations of silver nitrate solution were assessed by in vitro assays with Escherichia coli and MC3T3-E1, respectively. The ability of the silver-containing scaffolds to induce bacteriostasis was confirmed by the inhibition zone(15 mm) and high bactericidal rate([99 %). Cell proliferation, morphology and the alkaline phosphatase activity of MC3T3-E1 cultured on the scaffold with low silver phosphate contents were comparable with those cultured on control samples.

4-Axis printing microfibrous tubular scaffold and tracheal cartilage application

Long-segment defects remain a major problem in clinical treatment of tubular tissue reconstruction.The design of tubular scaffold with desired structure and functional properties suitable for tubular tissue regeneration remains a great challenge in regenerative medicine.Here,we present a reliable method to rapidly fabricate tissueengineered tubular scaffold with hierarchical structure via 4-axis printing system.The fabrication process can be adapted to various biomaterials including hydrogels,thermoplastic materials and thermosetting materials.Using polycaprolactone(PCL)as an example,we successfully fabricated the scaffolds with tunable tubular architecture,controllable mesh structure,radial elasticity,good flexibility,and luminal patency.As a preliminary demonstration of the applications of this technology,we prepared a hybrid tubular scaffold via the combination of the 4-axis printed elastic poly(glycerol sebacate)(PGS)bio-spring and electrospun gelatin nanofibers.The scaffolds seeded with chondrocytes formed tubular mature cartilage-like tissue both via in vitro culture and subcutaneous implantation in the nude mouse,which showed great potential for tracheal cartilage reconstruction.