软骨细胞与骨髓基质细胞共培养体外构建软骨组织的实验研究

目的 探讨利用软骨细胞提供的软骨微环境诱导骨髓基质细胞(BMSC)在体外构建软骨组织的可行性.方法 将分离出的猪骨髓基质细胞和软骨细胞进行体外培养,收集软骨细胞培养上清液,作为骨髓基质细胞诱导液从第2代开始进行诱导分化.7 d后取出标本,免疫组织化学检测软骨特异性Ⅱ型胶原表达,RT-PCR检测Ⅱ型胶原和aggrecan的mRNA表达.体外分离培养的骨髓基质细胞与软骨细胞,扩增后两者以8∶2比例混匀,以5.0×107/ml的终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA)支架,以相同浓度的单纯软骨细胞和单纯BMSC以及20%上述浓度(1.0×107/ml)的单纯软骨细胞作为对照组.标本于8周后取材,行大体观察、湿重、蛋白多糖(GAGs)含量测定、组织学及免疫组化等相关检测.结果 经诱导后的骨髓基质细胞的Ⅱ型胶原免疫组化检测阳性,RT-PCR检测Ⅱ型胶原和aggrecan mRNA呈阳性表达.混合细胞组及阳性对照组体外培养8周后形成了单一成熟的软骨组织,并保持了支架材料的大小和形状,两组新生软骨在外观及组织学特征上也基本相同,免疫组化结果 表明两组均大量表达软骨特异性细胞外基质Ⅱ型胶原,共培养组的平均湿重和蛋白多糖(GAGs)含量均达到阳性对照组的70%以上.而单纯骨髓基质细胞组仅在局部形成了极少量幼稚的软骨样组织,且材料支架明显皱缩变形.低软骨细胞浓度组虽新生软骨湿重量能达阳性对照组的30%,但材料支架明显皱缩变形,仅在局部形成了不连续的软骨组织,新生软骨量明显少于共培养各组及阳性对照组.结论 软骨细胞能在一定程度上提供软骨形成的微环境,有效地诱导BMSC向软骨细胞分化,并在体外形成组织工程化的软骨组织.

Abstract：

Objective To investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes. Methods The BMSCs and chondrocytes were separated from pig and cultured. The supernatant of chondrocytes was used as the inducing solution for BMSCs from the 2nd generation. 7 days later, samples were taken and underwent immunohistochemistry and RT-PCR for detection of the expression of specific type Ⅱ cartilage collagen,type Ⅱ collagen and aggrecan mRNA. The cultured BMSCs and chondrocytes were mixed at a ratio of 8:2(BMSC: cartilage cell) and were inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold at the final concentration of 5.0 × 107/ml. The cartilage cells and BMSCs were also inoculated seperately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the abovementioned concentration (1.0 × 107/ml) were used as the low concentration cartilage cell control group. Samples were collected 8 weeks later. General observations, wet weight, glycosaminoglycans (GAGs) determination and histological and immunohistochemistry examinations were performed. Results The expression of type Ⅱ collagen, type Ⅱ collagen and aggrecan mRNA were positive in induced BMSCs.In the co-cultured group and the positive control group, pure mature cartilage was formed after 8 weeks of culture in vitro, and the size and shape of the scaffold were maintained. The newly formed cartilage in the two groups were almost the same in appearance and histological properties. The immunohistochemistry results indicated that the cartilage cells of the two groups all expressed ample cartilage-specific type Ⅱ collagen. The average wet weight and GAG content in the co-cultured group reached more than 70% of those in positive control group. Only an extremely small amount of immature cartilage tissues was formed in local regions in pure BMSC group, and the scaffold was obviously shrunk and deformed. Although the wet weight of newly generated cartilage tissue in the low concentration cartilage cell group reached 30% of that in positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly formed cartilage was obviously less than that in the co-culture group and the positive control group. Conclusions Chondrocytes can provide a micro-environment for the formation of cartilage, and also effectively induce BMSC to differentiate into chondrocytes and form tissue-engineered cartilage in vitro.     

软骨细胞与骨髓基质细胞共培养体外构建软骨组织的实验研究

Objective To investigate the feasibility of chondrogenesis in vitro with bone marrow stromal cells (BMSCs) induced by the co-cultured chondrocytes. Methods The BMSCs and chondrocytes were separated from pig and cultured. The supernatant of chondrocytes was used as the inducing solution for BMSCs from the 2nd generation. 7 days later, samples were taken and underwent immunohistochemistry and RT-PCR for detection of the expression of specific type Ⅱ cartilage collagen,type Ⅱ collagen and aggrecan mRNA. The cultured BMSCs and chondrocytes were mixed at a ratio of 8:2(BMSC: cartilage cell) and were inoculated into a polyglycolic acid/polylactic acid (PGA/PLA) scaffold at the final concentration of 5.0 × 107/ml. The cartilage cells and BMSCs were also inoculated seperately at the same concentration as the positive and negative control. Pure cartilage cells at 20% of the abovementioned concentration (1.0 × 107/ml) were used as the low concentration cartilage cell control group. Samples were collected 8 weeks later. General observations, wet weight, glycosaminoglycans (GAGs) determination and histological and immunohistochemistry examinations were performed. Results The expression of type Ⅱ collagen, type Ⅱ collagen and aggrecan mRNA were positive in induced BMSCs.In the co-cultured group and the positive control group, pure mature cartilage was formed after 8 weeks of culture in vitro, and the size and shape of the scaffold were maintained. The newly formed cartilage in the two groups were almost the same in appearance and histological properties. The immunohistochemistry results indicated that the cartilage cells of the two groups all expressed ample cartilage-specific type Ⅱ collagen. The average wet weight and GAG content in the co-cultured group reached more than 70% of those in positive control group. Only an extremely small amount of immature cartilage tissues was formed in local regions in pure BMSC group, and the scaffold was obviously shrunk and deformed. Although the wet weight of newly generated cartilage tissue in the low concentration cartilage cell group reached 30% of that in positive control group, the scaffold was obviously shrunken and deformed. Only regional and discontinuous cartilage tissues were formed, and the amount of newly formed cartilage was obviously less than that in the co-culture group and the positive control group. Conclusions Chondrocytes can provide a micro-environment for the formation of cartilage, and also effectively induce BMSC to differentiate into chondrocytes and form tissue-engineered cartilage in vitro.     

软骨细胞与骨髓基质细胞共培养裸鼠体内构建软骨组织的实验研究

目的：探讨软骨细胞在裸鼠体内促进骨髓基质细胞（BMSCs）向软骨分化并形成软骨组织的可行性。方法：从SD大鼠中分别分离出BMSC和软骨细胞进行体外培养。收集软骨细胞培养上清液,作为BMSCs诱导液从第2代开始进行诱导分化,7天后取出标本,免疫组织化学检测软骨特异性Ⅱ型胶原表达,RT-PCR检测Ⅱ型胶原和aggrecan的mRNA表达。SD大鼠BMSCs与软骨细胞按一定比例（7：3）混匀,取5.0×107个混合细胞/ml的各组细胞悬液接种至壳聚糖生物材料,体外培养一周后植入裸鼠皮下,相同数量的单纯软骨细胞或BMSCs同样方法植入,分别作为阳性对照及阴性对照,1.5×107个软骨细胞同样植入作为低浓度软骨细胞对照。各组均8周后取材检测。结果：经诱导后的大鼠BMSCs的Ⅱ型胶原免疫组化检测阳性,RT-PCR检测Ⅱ型胶原和aggrecanmRNA呈阳性表达;混合细胞组及阳性对照组均形成了成熟的软骨,组织学可见成熟软骨陷窝、异染基质及Ⅱ型胶原表达;BMSCs组仅形成了纤维性组织;低浓度软骨细胞组在局部形成了少量软骨。结论：软骨细胞能在一定程度上提供软骨形成的微环境,诱导BMSCs在裸鼠体内向软骨组织分化并形成软骨组织。 还原     

软骨细胞与脂肪基质细胞共培养体外构建软骨的初步研究

目的 探讨软骨细胞与脂肪基质细胞(adipose-derived stromal cells,ADSCs)共培养体外构建软骨的可行性,并阐明软骨细胞提供的软骨微环境能否诱导ADSCs向软骨细胞分化并形成软骨组织.方法 分别培养扩增人ADSCs与猪耳软骨细胞,将2种细胞按7:3(ADSCs:软骨细胞)比例混匀,以5.0×107/ml的细胞终浓度接种于聚羟基乙酸/聚乳酸(PGA/PLA,直径8 mm,高2 mm)支架作为共培养组,以相同终浓度的单纯软骨细胞和单纯ADSCs分别接种相同支架作为阳性对照组及阴性对照组,以30％上述浓度(1.5×107/ml)的单纯软骨细胞接种作为低浓度软骨细胞对照组.每组各接种6例标本,每例接种细胞悬液200μl.全部标本均于体外培养8周时取材,通过大体观察、组织学、免疫组化及湿重、蛋白多糖定量检测等方法对新生软骨进行初步评价.多样本t检验统计分析各组湿重及蛋白多糖含量差异.结果 各组细胞均与材料粘附良好.共培养组及阳性对照组体外培养8周时基本保持了复合物初始大小和形状,大体观察2组均形成了较成熟软骨组织,组织学显示大量软骨基质和软骨陷窝形成,免疫组化显示软骨特异性细胞外基质Ⅱ型胶原分泌.定量测定结果表明,共培养组的平均湿重为(174±12) mg,平均蛋白多糖含量为(7.6±0.4) mg,两者分别达到阳性对照组的75％ (P＜ 0.01)和79％ (P＜ 0.01).阴性对照组(单纯ADSCs组)明显皱缩变形,组织学未见成熟软骨陷窝.低浓度软骨细胞组明显变薄,新生软骨平均湿重为(85 ±5) mg,是阳性对照组的37％ (P＜ 0.01),只在局部形成了不连续的软骨组织.结论 软骨细胞与ADSCs共培养能够在体外构建较成熟的软骨组织,软骨细胞能够诱导ADSCs成软骨分化及体外形成软骨组织.     

力学刺激促进软骨细胞与骨髓基质干细胞共培养体外软骨分化

目的：研究不同的应力刺激对软骨细胞与骨髓基质干细胞（BMSCs）共培养体外构建组织工程化软骨的影响。方法：分离、培养、扩传兔MSCs及软骨细胞,二者按7：3比例混和,以5．0×107／ml的细胞密度接种于聚羟基乙酸（PGA）支架上,一周后根据不同的施加力分为4组：离心组、摇床组、搅拌组,静止培养作为对照组。6周后取材行相关检测。结果：三受力组形成的细胞材料复合物基本保持原来的体积与外形。HE染色结果显示大量成熟软骨陷窝形成,细胞外基质沉积均匀;Safranin-O及甲苯胺兰染色显示有大量的GAG形成,免疫组化检测II型胶原表达强阳性。三受力组标本组织湿重、体积、GAG含量等指标均优于对照组。结论：力学刺激有利于促进少量软骨细胞与BMSCs共培养体外软骨分化;并在三维支架材料上构建组织工程化软骨。     

软骨细胞外基质源性取向支架与骨髓基质干细胞体外软骨组织工程的初步研究

目的 制备关节软骨细胞外基质源性取向支架,观察其对体外培养的骨髓基质干细胞分布排列的影响,探索其用于修复关节软骨缺损的可行性.方法 收集天然猪关节软骨,在PBS溶液中超微湿法粉碎关节软骨,利用差速离心收集细胞外基质悬液;低温超速离心收集沉淀,制备成2%～3%悬液;采用定向结晶与冷冻干燥技术制备取向支架,应用紫外交联及碳化二亚胺交联.光学显微镜及扫描电镜观察支架的形态结构,冰冻切片后组织化学染色对支架进行定性分析;生物力学方法检测支架的力学特性.分离培养兔骨髓基质干细胞,PKH26标记,接种到支架上体外软骨诱导培养,倒置荧光显微镜及扫描电镜观察培养3 d内种子细胞在支架内的黏附、分布及排列方式.结果 制备的支架材料具有垂直取向排列的孔道结构,软骨细胞外基质特异性染色阳性,纵向压缩弹性模量为(2.02±0.02)MPa,横向压缩弹性模量为(0.264±0.16)MPa,具有各向异性的力学特点.体外培养显示骨髓基质干细胞广泛均匀地分布在支架内部,并且在支架材料表层呈平行排列,深层呈柱状排列,类似于天然软骨组织中细胞的排列方式.结论 以关节软骨细胞外基质材料制备的取向性组织工程支架,在生化组成和结构上仿生天然关节软骨细胞外基质,是一种较为理想的软骨组织工程支架.     

同种异体软骨细胞与自体骨髓基质干细胞混合共培养构建软骨皮下移植的可行性研究

背景：软骨组织工程的种子细胞问题是目前研究的热点和难点,如何找到一种既能够避免对自体软骨进行取材又能够达到稳定软骨构建目的的方法呢？本研究尝试利用少量同种异体羊软骨细胞作为软骨诱导微环境提供者,与扩增后的羊自体BMSC混合共培养并植入皮下环境,探讨利用同种异体软骨细胞共培养构建软骨皮下移植的可行性。方法：本实验对山羊软骨细胞和BMSC分别进行取材和分离培养扩增,并将以上细胞分为以下四组进行混合并接种在PGA支架材料上：A组：100%自体软骨细胞;B组：30%自体软骨细胞＋70%自体BMSCs;C组：30%同种异体软骨细胞＋70%自体BMSCs;D组：100%同种异体软骨细胞。经过体外构建6周后植入羊皮下进行体内构建12周,对所形成的组织块进行大体观察和组织学染色等评价。结果：自体软骨细胞组和自体软骨细胞混合自体BMSC组皮下移植后可见成熟软骨组织形成,但同种异体软骨细胞参与的两组（包括同种异体软骨细胞混合自体BMSC的实验组和单纯异体软骨细胞组）在皮下环境中都因为较强的免疫反应未能形成软骨组织。结论：同种异体软骨细胞以及PGA支架材料的存在对于组织工程软骨在羊皮下环境的构建有负面影响。     

软骨细胞外基质源性取向支架与骨髓基质干细胞体外软骨组织工程的初步研究

Objective To fabricate cartilage extracellular matrix (ECM) oriented scaffolds and investigate the attachment, proliferation, distribution and orientation of bone marrow mesenchymal stem cells (BMSCs) cultured within the scaffolds in vitro. Methods Cartilage slices were shattered in sterile phosphate-buffered saline (PBS) and the suspension were differentially centrifugated untill the micro- fiber of the cartilage extracellular matrix was disassociated from the residue cartilage fragments. At last the supernatant were centrifugated, the precipitation were collected and were made into 2%-3% suspension. Using unidirectional solidification as a freezing process and freeze-dried method, the cartilage extracellular matrix derived oriented scaffolds was fabricated. The scaffolds were then cross-linked by exposure to ultraviolet radiation and immersion in a carbodiimide solution. By light microscope and scan electron microscope (SEM) observation, histological staining, and biomechanical test, the traits of scaffolds were studied. After being labelled with PKH26 fluorescent dye, rabbit BMSCs were seeded onto the scaffolds. The attachment, proliferation and differentiation of the cells were analyzed using inverted fluorescent microscope. Results The histological staining showed that toluidine blue, safranin O, alcian blue and anti-collagen Ⅱ immunohistochemistry staining of the scaffolds were positive. A perpendicular pore-channel structures which has a diameter of 100 μm were verified by light microscope and SEM analysis. The cell-free scaffolds showed the compression moduli were (2.02±0.02) MPa in the mechanical testing. Inverted fluorescent microscope showed that most of the cells attached to the scaffold. Cells were found to be widely distributed within the scaffold, which acted as a columnar arrangement. The formation of a surface cells layer was found on the surface of the scaffolds which resembled natural cartilage. Coclusion The cartilage extracellular matrix derived oriented scaffolds have promising biological, structural, and mechanical properties.     

骨髓基质细胞与关节软骨细胞生物学特性的比较研究

目的观察兔骨髓基质细胞(MSCs)诱导和基因修饰后的主要生物学特性,并与关节软骨细胞进行比较. 方法抽取成年雄性新西兰大白兔髂骨骨髓,密度梯度离心获得骨髓基质细胞,培养传至第5代,按处理方法分为常规培养液组(A组)、条件培养液组(B组)及重组缺陷型腺病毒携带肝细胞生长因子cDNA转染组(C组).条件培养液为常规培养液中含转化生长因子-β1(10 ng/ml)、碱性成纤维细胞生长因子(25 ng/ml)和地塞米松(10-7 mol/L).切取兔膝关节软骨,3 mg/ml Ⅱ型胶原酶消化传代培养至第3代(D组).观察原代MSCs及第5代MSCs(体外培养8～10周后)细胞形态,对第5代MSCs及第3代软骨细胞进行Ⅰ、Ⅱ型胶原免疫组织化学染色,MTT法检测细胞增殖情况.阿利新蓝法检测细胞培养上清液中糖胺多糖(GAG)含量.提取各组培养细胞总RNA,RT-PCR检测Ⅰ、Ⅱ型胶原表达. 结果原代MSCs为短梭形、簇状生长,传代细胞呈长梭形、旋涡样生长.A组细胞爬片Ⅰ型胶原免疫组织化学染色阳性,Ⅱ型胶原免疫组织化学染色阴性,GAG含量低,与D组比较,差异有统计学意义(P＜0.05).B组细胞爬片Ⅰ、Ⅱ型胶原免疫组织化学染色阳性,GAG含量升高,与D组比较差异无统计学意义(P＞0.05);C组转染后第4天增殖率降低,与A组比较差异有统计学意义(P＜0.05),其余时间点各组间无统计学意义(P＞0.05).RT-PCR表明A、B、C组均表达Ⅰ型胶原,B、D组可表达Ⅱ型胶原,C组有较弱的Ⅱ型胶原表达. 结论 MSCs体外培养过程中自然转归趋向于成骨.传代后经向成软骨方向诱导,具有向软骨分化的能力;体外传代培养的MSCs具有干细胞自我增殖和定向分化的特性,可作为靶细胞接受外源目的基因转染并能有效表达.     

经诱导的骨髓基质干细胞应用于组织工程化人工神经的实验研究

目的 探讨骨髓基质干细胞应用于组织工程化人工神经修复大鼠10mm长坐骨神经缺损的效果。方法 28只体重在160～200g的雌性F344大鼠随机分成4组，每组7只。A组：种植经诱导5d后的同源骨髓基质干细胞并具有内部支架结构的中空管；B组：种植同源许旺细胞并具有内部支架结构的中空管；C组：无细胞只具有内部支架结构的中空管；D组：自体神经移植组。术后3个月，进行系列神经电生理监测、坐骨神经功能指数测定、神经组织学观察、S—100及神经微丝蛋白兔疫组化染色和轴突计数等检查。结果 术后12周内，实验组(A组)的各项检测指标均优于C组(P＜0．05或0．01)，与B和D组间差异无显著性(P＞0．05)。结论 初步结果显示经诱导的骨髓基质干细胞可作为外周神经组织工程中的种子细胞，并应用于人工神经修复外周神经缺损。     

软骨细胞诱导骨髓基质干细胞构建带内支撑的组织工程化软骨

目的探讨以聚羟基乙酸(PGA)包裹特定形态的医用假体材料多孔高密度聚乙烯(HDPE,商品名为MEDPOR)为支架,应用软骨细胞诱导骨髓基质干细胞(BMSCs),共培养构建特定形态的带内支撑组织工程化软骨医用假体的可能性。方法以直径3mm、长5mm的圆柱形HDPE,外裹1mm厚PGA为支架,将体外分别培养的新生猪BMSCs和耳郭软骨细胞按7:3混合,以10×107/ml细胞浓度接种于支架上,同时以相同浓度的单纯软骨细胞和单纯BMSCs分别接种,作为阳性对照组(PC组)和阴性对照组(NC组)。经体外培养2周及在裸鼠皮下移植4、8周后取材,行大体观察、组织学、组织化学及免疫组化检测。结果各组细胞均与材料黏附良好。实验组和阳性对照组均形成了大体形态良好的HDPE-软骨复合体,内支撑的HDPE与外层软骨结合紧密。组织学可见成熟的软骨陷窝结构,软骨渗入HDPE孔隙内部、异染基质及Ⅱ型胶原呈强阳性表达。结论以HDPE为内支撑,外裹PGA的支架,接种混合细胞,可于皮下构建特定形态、组织学良好的HDPE-软骨复合体。     

自体骨髓基质干细胞在齿槽裂骨缺损修复中的应用

目的探讨人自体骨髓基质干细胞（human bone marrow stromal cells，hBMSCs）在治疗齿槽裂骨缺损中的可行性。方法2002至2005年，选择齿槽裂骨缺损患者7例（单侧6例，双侧1例），以患者自体骨髓基质干细胞为种子细胞，部分脱钙骨（partly demineralized bone matrix，pDBM）为支架材料构建组织工程骨，治疗齿槽裂骨缺损。从患者髂前上棘穿刺取骨髓，密度梯度离心法分离hBMSCs，经体外成骨诱导和扩增至第3代。将诱导的hBMSCs，复合部分脱钙骨体外培养1周后，手术回植骨缺损区。分别于术后1、3、6、12、24、36个月进行临床外形和三维CT检查随访。结果6例患者头部三维CT检查，结果示术后3个月能形成组织工程化骨，并修复骨组织缺损。术后1～3年的随访表明组织工程骨稳定存在，无明显骨吸收现象，临床治疗效果稳定。1例患者（双侧齿槽裂）植入物外露感染。结论以自体hBMSCs为种子细胞，部分脱钙骨为支架材料，利用组织工程技术可在人体内形成稳定的组织工程化骨组织，并临床修复齿槽裂骨缺损。     

联合培养时诱导的内皮细胞对自体骨髓基质干细胞成骨作用的影响

目的探讨由骨髓基质干细胞(BMSCs)诱导的内皮细胞(EC)与自体BMSCs共培养时对BMSCs成骨作用的影响。方法采用密度梯度离心法分离兔BMSCs,培养细胞分为四组：A组(BMSCs组)、B组(BMSCs成骨诱导组)、C组(EC诱导组)及D组(BMSCs和诱导的EC联合培养组),通过干细胞形态、免疫荧光、细胞增殖、碱性磷酸酶活性及骨钙素含量从酶学、组织学及生化等不同方面观察诱导的EC对BM- SCs成骨活性及牛长情况的影响。结果细胞免疫荧光染色证实C组培养诱导的细胞为EC。倒置相差显微镜、HE染色均显示EC与BMSCs混合生长良好。MTF检测结果：各组细胞增殖差异无显著性意义(P＞0．05)。碱性磷酸酶活性和骨钙素含量检测结果：D组明显高于其它各组,差异有显著性意义(P＜0．05)。结论由BMSCs诱导的EC能够增强BMSCs的成骨活性,提高BMSCs的增殖能力。     

骨髓基质细胞转化为神经细胞的研究

目的 探讨骨髓基质细胞体外转化为神经细胞的可能性。方法 骨髓基质细胞原代培养、传代后使用3种不同方法诱导。行大体观察，免疫组织化学，及细胞电生理检查和长期培养研究。结果 骨髓基质细胞体外可诱导为突触明确的神经细胞。诱导率分别为80％、10％和40％；诱导后细胞表面有神经特异性抗原NSE、GFAP表达；同时具有早期神经细胞电流特性。结论 骨髓基质细胞可横向转化为早期神经细胞。     

Effects of extracorporeal shock waves on human articular chondrocytes and ovine bone marrow stromal cells in vitro

Introduction We investigated the effects of extracorporeal shock waves on cytotoxicity and on the proliferation of human chondrocytes and ovine bone marrow stromal cells.Materials and methods Isolated cells were cultured to confluence, and 500 shock waves were applied at energy flux densities of 0, 0.02, 0.06, and 0.17 mJ/mm² for the cytotoxicity assay. The same energies at 100, 500, and 1000 impulses were used for the proliferation assay.Results Although bone marrow stromal cells revealed a dose- and impulse-dependent increase in the proliferation rate, no significant differences were found. Chondrocytes had less proliferative potential than untreated control groups. In the experimental set-up using 1000 impulses, proliferation was even higher in the control group. Both types of cells revealed a dose-dependent increase in cytotoxicity in the lactate dehydrogenase (LDH) assay.Conclusion As femoral head necrosis, osteochondritis dissecans, and similar disorders are increasingly treated with shock waves, their effect on human cartilage and chondrocytes deserves attention. We recommend further in vitro experiments with bone marrow stromal cells, as the latter might play an important role in the presumed multifactorial osteogenetic mechanism of shock waves due to their pluripotent character.The authors declare that the experiments comply with the current laws.