Effect of rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins in vitro

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation because it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates and a calcium phosphate bone cement on the expression of bone-related genes and proteins by human bone-derived cells (HBDCs) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, two materials with a crystalline phase Ca(2)KNa(PO(4))(2) and with a small amorphous portion containing either magnesium potassium phosphate (material denominated GB14) or silica phosphate (material denominated GB9), and a calcium phosphate bone cement (material denominated Biocement D). HBDCs were grown on the substrata for 3, 7, 14, and 21 days, counted, and probed for various mRNAs and proteins (type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase, and bone sialoprotein). All substrates supported continuous cellular growth for 21 days. In the presence of GB14 and Biocement D specimens cell proliferation was reduced and cell differentiation increased. At day 21, the greatest number of cells was found on GB9 expressing significantly higher levels of bone-related proteins than cells grown on all other surfaces. Because all novel materials facilitated the expression of the osteoblastic phenotype at least as much as TCP and the polystyrene control, these biomaterials can be regarded as excellent candidate bone substitute materials. GB9 induced the highest proliferation and cellular differentiation after 21 days of incubation, suggesting that this material may possess a higher potency for enhancing osteogenesis than TCP.     

Composites made of rapidly resorbable ceramics and poly(lactide) show adequate mechanical properties for use as bone substitute materials.

Porous composite materials made of poly(L, DL-lactide) and a ceramic component, alpha-tricalcium phosphate (alpha-TCP) or one of the rapidly resorbable glass ceramics, GB9N or GB14N, respectively, were developed to be used as bone substitutes. The present article describes the mechanical properties and the in vitro degradation characteristic of the different composite materials. The yield strength, the elastic modulus, and the molecular weight were measured after in vitro degradation up to 78 weeks. The initial strengths of the alpha-TCP composite (12.5 +/- 0.7 MPa) was higher than that of the GB9N and GB14N composites (8.3 +/- 0.2 MPa and 10.9 +/- 0.2 MPa, respectively). The initial elastic moduli of the composites were between 450 and 650 MPa. The mechanical properties remained constant until a degradation period of 26 weeks. Then they decreased continuously until they were completely lost at week 52. The molecular weight (M(w)) decreased steadily from 91,000 D in the case of the alpha-TCP composite and 78,000 D and 85,000 D in the case of the GB9N or GB14N composites, respectively, to about 10,000 D at week 78. It was concluded that the composites show adequate mechanical properties in the range of cancellous bone and a suitable degradation characteristic to be used as bone substitute materials.     

Histological and histomorphometric investigations on bone integration of rapidly resorbable calcium phosphate ceramics

Resorbable ceramics can promote the bony integration of implants. Their rate of degradation should ideally be synchronized with bone regeneration. We report here the results of a histological study of implants with two resorbable calcium phosphate ceramic coatings: Ca(2)KNa(PO(4))(2)-(GB14) and Ca(10)[K/Na](PO(4))(7)-(602020). The results attained with these ceramic-coated implants show the benefits of these materials with regard to bioactive bone-healing stimulation, compared with uncoated implants. The GB14 ceramic coating exhibited greater bone regeneration and differentiation on its surface than the conventional hydroxyapatite coating and helped bone tissue achieve more extensive contact free of connective tissue. Not until the coating disintegrated did the histological features of GB14- and 602020-coated implants converge-both implant types were integrated into bone. Rapid disintegration of the coating material, as with 602020, supports osteoblast proliferation but has negative effects on bone mineralization. Both resorbable ceramics tested, GB14 and 602020, demonstrated bioactivity; even metal surfaces coated with these materials were populated by mature bone tissue without connective tissue after disintegration of their ceramic coating. The less rapidly degrading material, GB14, achieved better results. Degradable calcium phosphate coatings have the potential to stimulate bone regeneration. From the histological viewpoint, the resorbable ceramics examined here can be recommended as coating materials for clinical use.     

Effects of 58S sol-gel glasses on the temporal expression of bone markers during mouse osteoblastic differentiation

Previous studies have shown that bioactive glasses can support osteoblastic growth and differentiation in vitro as well as in vivo. More recently, a new category of sol-gel glasses has been developed with enhanced bioactivity and open pores enclosed in a mesoporous matrix. In our study, we investigated the effect of 58S sol-gel glasses on the growth and differentiation of mouse calvaria osteoblasts. Two types of granules were used: 58S sol-gel granules and 60S inert glasses used as control. Phase contrast microscopy showed that cells proliferated and formed mineralized bone nodules in both cultures. However, this phenomenon occurred earlier and to a higher degree in cultures with 58S sol-gel glasses. Northern blot analysis of the expression of osteoblastic markers revealed that osteoblasts retained their phenotype in both types of cultures. Interestingly, stimulation of alkaline phosphatase, bone sialoprotein, and osteocalcin was noticed at day 18 in sol-gel cultures when compared with that in control. These data confirm that 58S bioactive glasses are capable of supporting the growth and maturation of primary mouse osteoblasts. In addition, it was shown that 58S glasses affected the gene-expression profile, causing an up-regulation of the major bone markers. These results indicated that 58S sol-gel glasses appeared as suitable candidates for osteoblast scaffolds in the field of bone tissue engineering.     

Evaluation of a resorbable, in situ setting bone substitute in a sheep model

The gold standard for bone substitution is the autologous bone graft, but because of its limited supply and the associated morbidity, the search for synthetic alternatives is necessary. A new in situ setting tricalcium phosphate cement was implanted in a trepanation defect (9.4 mm diameter, 10 mm depth) in the distal femoral epiphysis of sheep. Empty cavities and autologous bone graft were used as controls. Histologic and histomorphometric examinations were carried out after 12 weeks. Nearly 90% of the implanted cement was resorbed and replaced by ingrown bone with close contact between surrounding bone, new bone, and remaining cement particles. The amount of bone in the defect area was significantly higher in defects filled with cement relative to defects filled with autologous bone graft (mean 27 vs. 21%, 95% confidence intervals 23 to 31 and 18 to 23, p = 0.026). In conclusion, this new in situ setting cement is bioactive, resorbable, and osteoconductive. It will be useful as an alternative to autologous bone graft to fill stable defects.     

Effect of bone extracellular matrix synthesized in vitro on the osteoblastic differentiation of marrow stromal cells

Alternative materials for bone grafts are gaining greater importance in dentistry and orthopaedics, as the limitations of conventional methods become more apparent. We are investigating the generation of osteoinductive matrix in vitro by culturing cell/scaffold constructs for tissue engineering applications. The main strategy involves the use of a scaffold composed of titanium (Ti) fibers seeded with progenitor cells. In this study, we investigated the effect of extracellular matrix (ECM) laid down by osteoblastic cells on the differentiation of marrow stromal cells (MSCs) towards osteoblasts. Primary rat MSCs were harvested from bone marrow, cultured in dexamethasone containing medium and seeded directly onto the scaffolds. Constructs were grown in static culture for 12 days and then decellularized by rapid freeze-thaw cycling. Decellularized scaffolds were re-seeded with pre-cultured MSCs at a density of 2.5 x 10(5) cells/construct and osteogenicity was determined according to DNA, alkaline phosphatase, calcium and osteopontin analysis. DNA content was higher for cells grown on decellularized scaffolds with a maximum content of about 1.3 x 10(6) cells/construct. Calcium was deposited at a greater rate by cells grown on decellularized scaffolds than the constructs with only one seeding on day-16. The Ti/MSC constructs showed negligible calcium content by day-16, compared with 213.2 (+/- 13.6) microg/construct for the Ti/ECM/MSC constructs cultured without any osteogenic supplements after 16 days. These results indicate that bone-like ECM synthesized in vitro can enhance the osteoblastic differentiation of MSCs.     

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.     

Effects of in vitro bone formation on the mechanical properties of a trabeculated hydroxyapatite bone substitute

This study was designed to test the hypothesis that the mechanical properties of a trabecular bone substitute can be enhanced through in vitro tissue formation. Our specific objectives were to (1) determine the effects of in vitro marrow stromal cell-mediated tissue deposition upon a trabeculated hydroxyapatite scaffold on the strength and toughness of the resulting bone substitute; and (2) identify and characterize regions of newly deposited matrix and mineral. This work provides a basis for future investigations aimed at transforming a brittle hydroxyapatite scaffold into an osteoinductive, biomechanically functional implant through in vitro bone deposition. As hypothesized, the mechanical properties of the trabecular bone substitutes were significantly enhanced by in vitro tissue formation. As a result of cell seeding and a 5 week culture protocol, mean strength increased by 85% (p = 0.008) and energy to fracture increased by 130% (p = 0.003). Accompanying the enhancement of mechanical properties was the deposition of significant amounts of bone matrix and mineral. Fluorescence imaging, scanning electron microscopy, electron probe microanalysis, and nanoindentation confirmed the presence of bonelike mineral with Ca/P ratio, modulus, and hardness similar to that within human and rat trabecular bone tissue. This new mineralization was found to exist within a newly deposited parallel-fibered matrix both encasing and bridging between scaffold trabeculae. Taken as a whole, our results establish the feasibility of the production of an osteoinductive hydroxyapatite-based trabecular bone substitute with mechanical properties enhanced through in vitro bone deposition.     

骨修复替代材料修复骨缺损的选择与应用

背景：在骨缺损治疗方面,近几年集中在骨修复替代材料领域出现了一系列重大科研突破,催生了很多全新的骨修复替代材料。目前临床上可供选择的骨修复替代材料种类繁多,各种材料的特性不同,各有优势和缺点。如何正确选择与应用是创伤骨科医师面临的实际问题,值得讨论和推敲。 目的：通过对自体骨、同种异体骨和人工骨材料在应用中的相关文献分析,评价3种骨修复替代材料的生物性能,有利于骨科医师更好的理解几种骨修复替代材料的理化、生物性能,进一步提高诊断和治疗水平。 方法：人工骨材料在骨缺损修复中起到不可取代的作用,并且也获得良好的临床效果,但近期临床效果并非是与传统材料的最终比较结果。人工骨材料安全性和稳定性的验证尚缺乏足够的实验依据,确切疗效尚须长期的临床观察。寻找具有良好生物特性的人工骨材料抑制是骨科的研究热点。分别对自体骨、同种异体骨、人工骨材料3种骨修复替代材料治疗骨缺损的理化、生物性能,安全性综合比较进行实验数据分析。 结果与结论：①在骨缺损修复过程中,从修复质量、免疫排斥和疾病传播等多方面来衡量,自体骨都是最佳的选择,成为骨移植的金标准。但来源有限且取骨区可能产生并发症,取骨又造成第二术区的创伤,因而它的临床应用受到了很大的限制。②同种异体骨的来源比自体骨多,但与宿主间的免疫排斥反应,并有感染疾病如肝炎病毒的可能。③人工骨材料成型迅速,在计算机辅助设计和辅助制作的帮助下,能更好的合成与缺损骨形态接近的骨修复替代材料,人工骨材料的骨诱导性和仿生物性骨结构方面还不能令人满意。④现有的骨修复替代材料均存在各自不同某些缺陷,不能完全满足临床质量的需要。未来能代替天然骨骼,解决骨缺损修复的材料应当是优势的组合,是骨修复替代材料的发展趋势。     

The effect of starch and starch-bioactive glass composite microparticles on the adhesion and expression of the osteoblastic phenotype of a bone cell line

There is a clear need for the development of microparticles that can be used simultaneously as carriers of stem/progenitor cells and as release systems for bioactive agents, such as growth factors or differentiation agents. In addition, when thinking on bone-tissue-engineering applications, it would be very useful if these microparticles are biodegradable and could be made to be bioactive. Microparticles with all those characteristics could be cultured together with adherent cells in appropriate bioreactors to form in vitro constructs that can then be used in tissue-engineering therapies. In this work, we have characterized the response of MC3T3-E1 pre-osteoblast cells to starch-based microparticles. We evaluated the adhesion, proliferation, expression of osteoblastic markers and mineralization of cells cultured at their surface. The results clearly show that MC3T3-E1 pre-osteoblast cells adhere to the surface of both polymeric and composite starch-based microparticles and express the typical osteoblastic marker genes. Furthermore, the cells were found to mineralize the extracellular matrix (ECM) during the culture period. The obtained results indicate that starch-based microparticles, known already to be biodegradable, bioactive and able to be used as carriers for controlled release applications, can simultaneously be used as carriers for cells. Consequently, they can be used as templates for forming hybrid constructs aiming to be applied in bone-tissue-engineering applications.     

In vitro biocompatibility of resorbable experimental glass ceramics for bone substitutes

Tricalcium phosphate ceramics (TCPs) are increasingly used as bone substitutes. They demonstrate good biocompatibility and degrade relatively slowly. New glass ceramics based on calcium alkali orthophosphates (Ca(2)KNa(PO(4))(2)) were developed that degrade faster than TCP but could have reduced biocompatibility due to their high solubility. Therefore, they were modified by a neutralizing surface treatment. The aim of this study was to evaluate the biocompatibility of some of these ceramics, GB1a, GB9, and GB14, which differ in the amount of added Na, K, Mg, or Si ions, with standard and modified surfaces. The in vitro cytotoxicity of the ceramics GB1a, GB9, and GB14 was determined by the agar diffusion and filter test and the microculture tetrazolium (MTT) assay. In order to investigate the influence of surface modification, these three ceramics were compared to their surface-treated counterparts, GB1aN, GB9N, and GB14N. GB1a, the ceramic with the highest in vitro solubility, showed the strongest toxic influence in all cell culture tests. GB9 and GB14 produced better results. In contrast, the counterparts with modified surfaces exhibited no (GB9N, GB14N) or weak (GB1aN) signs of cytotoxicity. It is concluded that the toxicity of the ceramics GB1a, GB9, and GB14 depends on their solubility. A positive influence of the surface treatment on in vitro biocompatibility was demonstrated. Therefore, the surface-treated glass ceramics could be promising materials for bone replacement.     

Effect of enamel matrix proteins on the phenotype expression of periodontal ligament cells cultured on dental materials

Cells within the periodontal ligament have the potential to regenerate a periodontal connective tissue attachment on pathologically exposed root surfaces as well as on several material surfaces including titanium. However, rather than a periodontal connective tissue attachment, a fibrous encapsulation or chronic inflammatory response has been reported at the material connective tissue interface for most dental materials. Cementum is the first tissue of the periodontal connective tissue attachment to develop and the secretion of enamel matrix related proteins on the newly mineralized dentin surface precedes and is thought to induce cementum formation. Enamel matrix-related proteins may also function in the adult because the application of an acid extract of porcine enamel protein matrix (Emdogain(R), EMD) on pathologically exposed root surfaces has been shown to result in cementum regeneration. Therefore, the objective of the present study was to determine whether the application of EMD to materials that do not normally support cementogenesis in vivo would alter the in vitro phenotype of periodontal ligament (PDL) cells including the synthesis of cementum-associated extracellular matrix proteins. Primary PDL cells were established from 21-day-old Sprague-Dawley rats, and were cultured on four materials commonly encountered in dental practice (gutta percha, calcium hydroxide, amalgam, and super EBA cement) with and without the application of EMD. After 7 or 14 days of culture, total-DNA content, collagen synthesis, alkaline phosphatase activity, and the synthesis of a 42-kDa cementum-associated extracellular matrix protein were determined. PDL cells cultured on all materials had decreased total DNA content. The application of EMD further decreased total DNA content. PDL cells cultured on gutta percha and calcium hydroxide with the application of EMD had similar levels of collagen synthesis and alkaline phosphatase activity but also expressed a 42-kDa cementum extracellular matrix-associated protein when compared to the other groups. These results suggest that EMD can alter the phenotype of PDL cells when cultured on these dental materials.     

Application of perfusion culture system improves in vitro and in vivo osteogenesis of bone marrow-derived osteoblastic cells in porous ceramic materials

Composites of bone marrow-derived osteoblasts (BMOs) and porous ceramics have been widely used as a bone graft model for bone tissue engineering. Perfusion culture has potential utility for many cell types in three-dimensional (3D) culture. Our hypothesis was that perfusion of medium would increase the cell viability and biosynthetic activity of BMOs in porous ceramic materials, which would be revealed by increased levels of alkaline phosphate (ALP) activity and osteocalcin (OCN) and enhanced bone formation in vivo. For testing in vitro, BMO/beta-tricalcium phosphate composites were cultured in a perfusion container (Minucells and Minutissue, Bad Abbach, Germany) with fresh medium delivered at a rate of 2 mL/h by a peristaltic pump. The ALP activity and OCN content of composites were measured at the end of 1, 2, 3, and 4 weeks of subculture. For testing in vivo, after subculturing for 2 weeks, the composites were subcutaneously implanted into syngeneic rats. These implants were harvested 4 or 8 weeks later. The samples then underwent a biochemical analysis of ALP activity and OCN content and were observed by light microscopy. The levels of ALP activity and OCN in the composites were significantly higher in the perfusion group than in the control group (p < 0.01), both in vitro and in vivo. Histomorphometric analysis of the hematoxylin- and eosin-stained sections revealed a higher average ratio of bone to pore in BMO/beta-TCP composites of the perfusion group after implantation: 47.64 +/- 6.16 for the perfusion group and 26.22 +/- 4.84 for control at 4 weeks (n = 6, p < 0.01); 67.97 +/- 3.58 for the perfusion group and 47.39 +/- 4.10 for control at 8 weeks (n = 6, p < 0.05). These results show that the application of a perfusion culture system during the subculture of BMOs in a porous ceramic scaffold is beneficial to their osteogenesis. After differentiation culture in vitro with the perfusion culture system, the activity of the osteoblastic cells and the consequent bone formation in vivo were significantly enhanced. These results suggest that the perfusion culture system is a valuable and convenient tool for applications in tissue engineering, especially in the generation of artificial bone tissue.     

Enhanced expression of the osteoblastic phenotype on substrates that modulate fibronectin conformation and integrin receptor binding

Integrins represent the primary mechanism of cell-extracellular matrix interactions and control cell morphology, proliferation, and differentiation. We have previously shown that substrate-dependent modulation of adsorbed fibronectin (Fn) conformation alters alpha5beta1 integrin binding to Fn and directs C2C12 myoblast proliferation and differentiation (Mol. Biol. Cell 10 (1999) 785). The model substrates used in these experiments were bacteriological (untreated) polystyrene (B), tissue culture polystyrene (T), and type-I collagen-coated T (C). In the present study, we examined MC3T3-EI osteoblast-like cell differentiation on Fn-coated B, T, and C substrates. Immunofluorescence staining revealed substrate-dependent differences in integrin alpha5beta1 binding and clustering into focal adhesions (C > T > B), consistent with our previous integrin binding analysis. Alkaline phosphatase activity and matrix mineralization showed substrate-dependent differences (C > T > B, p < 0.05). Similar trends were observed for alkaline phosphatase, osteocalcin, and bone sialoprotein gene expression. Blocking experiments with antibodies directed against Fn completely inhibited matrix mineralization on Fn-coated C, indicating that Fn is critical to expression of the osteoblastic phenotype on this extracellular matrix component. These substrate-dependent differences in osteoblast differentiation correlated with differences in alpha5beta1 binding, suggesting that these differences arise from substrate modulation of integrin-matrix interactions. Substrate-dependent modulation of cell function may provide a versatile mechanism to control cell responses in numerous biomedical applications.     

Effect of degradation rates of resorbable phosphate invert glasses on in vitro osteoblast proliferation

Four resorbable phosphate invert glasses for use as bone replacement were synthesized in the system P2O5--CaO--MgO--Na2O. TiO2 and SiO2 were added at concentrations of 1 and 5.5 mol % to control solubility and crystallization. Both bulk glasses and samples with an open porosity of 65% and pore sizes of 150 to 400 microm were produced using a salt sintering process. Addition of TiO2 decreased the solubility in water and simulated body fluid, while the glass with addition of SiO2 showed a higher dissolution rate than did the original glass. The hypothesis that dissolution rates of the glasses will affect cell proliferation of osteoblastlike cells was tested using a MC3T3-E1.4 murine preosteoblast cell line. Cells were cultured on nonporous polished and porous glasses with tissue culture polystyrene (TCPS) as control. Cell proliferation was studied over 24 and 72 h in culture. Cells proliferated on all polished glasses, but proliferation on porous glasses showed variations with glass composition. Cell proliferation increased with decreased solubility of the glass. It is suggested that resorbable implant materials require the adjustment of dissolution rate so as to facilitate cell adhesion and proliferation and thus a gradual transition from artificial implant to new bone structure.     

Quantitative kinetic analysis of gene expression during human osteoblastic adhesion on orthopaedic materials

Little information was found in the literature about the expression on hydroxyapatite (HA) materials of genes specific of cellular adhesion molecules although more were found on titanium-based substrates. Hence, the goal of this work was to study by a kinetic approach from 30 min to 4 days the adhesion of Saos-2 cells on microporous (mHA) and non-microporous hydroxyapatite (pHA) in comparison to polished titanium. Our strategy associated the visualization of adhesion proteins inside the cells by immunohistochemistry and the quantitative expression of genes at mRNA level by real-time PCR. The cell morphology was assessed using scanning electron microscopy and the number of cells thanks to biochemical techniques. The cellular attachment was the highest on mHA from 30 min to 24 h although the cell growth on mHA was the lowest after 4 days. Generally, the Saos-2 osteoblastic cells morphology on mHA was radically different than on other surfaces with the particularity of the cytoplasmic edge, which appeared un-distinguishable from the surface. The revelation by specific antibodies of proteins of the cytoskeleton (actin) and the focal adhesions (FAK, phosphotyrosine) confirmed that adhesion and spreading were different on the 3 materials. The actin stress fibres were less numerous and shorter on mHA ceramics. Cells had more focal contacts after 4 h on mHA compared to other substrates but less after 24 h. The highest values of total proteins were extracted from mHA at 0.5 and 24 h and from pHA at 1, 4, and 96 h. The alphav and beta1 integrin, actin, FAK, and ERK gene expression were found to be different with adhesion time and with materials. C-jun expression was comparable on mHA, titanium and plastic but was largely higher than on pHA at 0.5 and 1 h. On the contrary, c-fos expression was the highest on pHA after 0.5 h and the lowest after 1h. This difference between c-fos and c-jun expression on pHA after 0.5 h could be related to the fact that these two genes may differ in their signalling pathways. The expression of the alkaline phosphatase gene after 4 days was lower on mHA compared to other materials demonstrating that the microstructure of the mHA ceramic was not favourable to Saos-2 cells differentiation. Finally, it was demonstrated in this study that HA and titanium surfaces influence as well gene expression at early times of adhesion as the synthesis of adhesion proteins but also proliferation and differentiation phases. Indeed, the signal transduction pathways involved in adhesion of Saos-2 cells on HA and titanium were confirmed by the sequential expression of alphav and beta1 integrins, FAK, and ERK genes followed by the expression of c-jun and c-fos genes for proliferation and alkaline phosphatase gene for differentiation.     

鹿茸多肽诱导骨髓间充质干细胞体外向软骨表型的分化

背景：实验证实鹿茸多肽可以促进体外培养软骨细胞的增殖和细胞外基质糖胺多糖、Ⅱ型胶原、Aggrecan蛋白的表达。 目的：通过对体外培养的兔骨髓间充质干细胞在特定培养液作用下向软骨细胞表型分化的研究,探讨鹿茸多肽对其软骨分化的影响。 方法：将第3代兔骨髓间充质干细胞随机分为空白对照组、诱导组、鹿茸多肽组,分别采用普通培养液、诱导培养液、含10 mg/L鹿茸多肽的诱导培养液于离心管内进行培养;并取兔的关节软骨细胞作为关节软骨组。分别于1,2,3周后取材,通过组织学、生物化学和RT-PCR技术,对离心管内构建的软骨组织进行形态学和细胞功能状态的观察。 结果与结论：空白对照组培养2周后,细胞团块逐渐崩解,无法进行苏木精-伊红染色。诱导组、鹿茸多肽组细胞团块除有轻度收缩外,呈白色半透明状;苏木精-伊红染色发现部分细胞为圆形或卵圆形,表层细胞密度大;诱导组、鹿茸多肽组糖胺多糖含量及Ⅱ型胶原mRNA表达随培养时间延长而增多,各时间点诱导组、鹿茸多肽组含量均高于空白对照组(P < 0.05);各时间点鹿茸多肽组糖胺多糖含量及Ⅱ型胶原mRNA表达均高于诱导组,但低于关节软骨组 (P< 0.05)。提示骨髓间充质干细胞在特定培养条件下能向软骨细胞表型分化,且鹿茸多肽对其定向软骨分化有明显促进作用。虽然在体外可以构建出软骨组织,但其与关节软骨质量相比仍有很大差距。