Differentiation of periosteal cells in muscle. An experimental study using the diffusion chamber method

The osteo-chondrogenic capacity of the undifferentiated mesenchymal cells of the periosteum has been made use of in clinical reconstructive surgery. In the present investigation we studied the osteo-chondrogenic potency of free periosteal transplants in muscle using the diffusion chamber method. A total of 42 experimental and seven control rabbits aged four to six weeks were operated on. Periosteum was obtained from the anterior aspect of the left tibial bone by stripping. The grafts were placed in Nucleopore diffusion chambers with a pore size of 0.4 micron. The chambers were implanted in the anterior tibial and paraspinal muscles of the rabbit. Osteogenesis began after the second postoperative week and increased up to the 5-6 week interval when a plateau phase was reached. Chondrogenesis, which also began after the second postoperative week, reached two plateau phases; the first observed at 4-8 weeks and the second at 12-16 weeks. Neither bone nor cartilage formation could be observed outside the chambers. In the semi-open control chambers with only one end sealed, bone formed within the chamber as early as two weeks after transplantation and grew out into the adjacent connective tissue of muscle. It is noteworthy that the periosteal transplant retained its osteochondrogenic properties even when isolated in the diffusion chamber. The young age of the donor animals might have contributed to our findings.     

Reconstruction of patellar cartilage defects with free periosteal grafts. An experimental study

The osteo-chondrogenic potential of free periosteal grafts was investigated within the knee joint in 26 rabbits aged four to six weeks. A total of 36 knee joints were operated on. The grafts were stripped from the medial side of the right tibia and sutured on the articular surface of the patella, from which the cartilage had been totally excised to the subchondral bone. In 16 knees the graft was sutured with the cambium layer towards the subchondral bone and in eight knees the fibrous layer faced the bone. In the control group of twelve knees the patellar articular cartilages were excised and no periosteal transplant was grafted to the patellar articular surface. In the transplantation group cartilage formation could be seen already one week after the operation. There were no marked differences between the series with the cambium layer facing the subchondral bone or the group with the fibrous layer facing the bone. At 20 weeks the hypertrophied cartilage had thinned and resembled normal joint cartilage. In the control group the histological picture resembled osteoarthritis.     

不同孔径β-TCP对大鼠骨髓间充质干细胞增殖及成骨作用的体外研究

目的 探讨不同孔径β-磷酸三钙(β-TCP)对大鼠骨髓间充质干细胞(BMSC)增殖及成骨分化的作用.方法 体外原代培养BMSC,取第2代细胞悬液100μl(细胞浓度2×106/ml)接种于5组不同孔径(187～300、300～375、375～500、500～750、750～830 μm)的圆盘状多孔 β-TCP材料中.采用细胞计数试剂盒(CCK-8)测定BMSC接种后3、24、48、72 h增殖情况;扫描电镜观察接种72 h后BMSC在多孔β-TCP上贴附、生长情况;碱性磷酸酶(ALP)试剂盒检测接种72 h后BMSC的ALP活性.结果 接种后48 h BMCS在各组多孔β-TCP上均有显著增殖,其中500～750 μm组增殖最快(P＜0.05);扫描电镜观察显示,BMCS在多孔β-TCP材料上黏附、伸展、聚集,生长状态良好;接种后72 h各组BMCS的ALP活性均明显增高,其中500～750 μm组ALP活性最高(P＜0.05).结论 不同孔径的多孔β-TCP对BMSC增殖及成骨分化均有促进作用,其中500～750 μm孔径的促进作用最明显,可能是促进BMSC增殖及成骨分化的最适孔径.     

骨髓间充质干细胞分化为血管内皮细胞的实验研究

目的：探讨骨髓间充质干细胞（MSCs)分化为肉芽组织中血管内皮细胞的可能性。及其参与创面修复的可能机制。方法：抽取小型香猪的骨髓，经密度梯度离心分离、纯化MSCs，体外培养扩增后，应用溴脱氧尿嘧啶（BrdU)标记技术标记细胞。于已抽取骨髓的小型香猪背部制作全层皮肤缺损创面，即刻以纤维蛋白胶为载体，将已标记的MSC回植到供体动物创面上。术后2、4、6、8、12周切取创面组织，行BrdU和因子Ⅷ（FⅧ）免疫组织化学染色，进行对比观察。结果：BrdU阳性的MSCs多聚集在创面肉芽组织中的小血管周围，且有个别血管内皮细胞也呈现BrdU 阳性。部分BrdU阳性的MSCs胞浆中亦有FⅧ表达。结论：创面愈合过程中，MSCs与肉芽组织中小血管的形成密切相关。在创面微环境下，MSCs可分化为血管内皮细胞，并参与创面修复。     

Behaviour of cancellous bone graft with and without periosteal isolation in striated muscle. An experimental study

The capacity of the periosteum to inhibit resorption of cancellous bone grafts into muscle was investigated in 34 four- to six-week-old rabbits. In 17 experiments the periosteum was wrapped around the grafts with the cambium layer facing the bone, and in seven experiments with the cambium layer facing the muscle. In the control group of 10 experiments there was no periosteal wrapping around the bone grafts. In Series 1 with the cambium layer of the periosteum facing the bone, after 20 weeks a tubular bone with Haversian system and bone marrow was seen. The transplants were surrounded by normal-looking periosteum. Bone formation from the periosteum occurred through enchondral ossification. Inductive bone growth was observed from the cancellous graft. In Series 2 with the cambium layer facing the surrounding muscle tissue, after 20 weeks two laminar bone blocks with periosteum in between and surrounding each block was observed. In the control series without periosteal covering, after 20 weeks only fibrous tissue remained in the transplantation site. It is obvious that periosteal isolation of cancellous bone grafts inhibits their resorption when transplanted into muscle in young animals.     

The effect of partial periosteal division on growth of the long bones. An experimental study in rabbits

The mode of action of the periosteum in the growth of the long bones was investigated by comparing the activity of the growth plate after hemicircumferential and longitudinal periosteal incisions. Twenty-eight rabbits were divided into three groups. A longitudinal periosteum incision was made on the medial upper tibia in rabbits of Group A, and a hemicircumferential periosteum incision was made in Group B. An incision of the skin and superficial tissue only, similar to the skin incision of Groups A and B, was performed on rabbits in Group C. Certain differences in the development of the right tibia compared to the control side were observed in rabbits of Group B: (1) valgus deformity, 5 degrees-10 degrees; (2) overgrowth, 1-2 mm; (3) an S-shaped tibia deformity. The dynamics of the deformity support the mechanical theory because the direction of periosteum division was an important factor in the appearance of growth disturbances.     

加味左归丸诱导骨髓间充质干细胞骨向分化的实验研究

目的 观察加味左归丸对体外培养的骨髓间充质干细胞(Mesenchymal Stem Cells,MSCs)向成骨细胞分化的影响。方法 采用全骨髓法（贴壁筛选法）培养大鼠MSCs,用加味左归丸含药血清诱导大鼠MSCs向成骨细胞分化,并进行形态学观察和免疫细胞化学技术检测。结果 与空白对照组相比,加味左归丸组与经典诱导组均可以明显促进成骨表型化MSCs的矿化结节形成及I型胶原表达（P<0.05）。结论 加味左归丸可明显促进大鼠MSCs向成骨细胞转化,优化骨组织工程的种子细胞体系,改善骨组织的质量,促进骨组织的再生。     

Use of revascularized periosteal allografts for repairing bony defects: An experimental study

This is an experimental study on the potential of revascularized periosteal allografts for repair of bony defects. Beijing long-eared white rabbits were used as donors and immunologically incompatible Chinchilla rabbits were used as recipients. The periosteum was stripped off from the femurs of the 6 donor rabbits and transplanted to bony defects of 1.2 cm over the radius of the 12 recipients using microsurgical anastomoses of the blood vessels. In the control group of 8 rabbits the same radial bony defects were produced without the transplantation of periosteal grafts. Immunosuppressants were administered after surgery. The animals were observed for 4 months using a series of radiography, histomorphology, fluochrome labelling and electron microscopy. Results revealed no bony healing of the defects in the control group, but 9 of the 12 defects healed in the experimental group. The results indicated revascularized periosteal allografts were able to form new bone and heal the 1.2 cm radial bone defect in rabbits. © 1994 Wiley-Liss, Inc.     

Evaluation of osteogenic/chondrogenic cellular proliferation and differentiation in the xenogeneic periosteal graft

To determine whether grafted young periosteum can induce new bone formation in elderly patients, this preliminary study evaluated cell proliferation and differentiation in xenogeneic periosteal grafts in old rats radiographically, histologically, and immunohistochemically. Periosteum harvested from the tibia of young Japanese white rabbits were grafted into old Sprague-Dawley rats with or without administration of 1.0 mg per kilogram per day immunosuppressant FK506. Autogenous old periosteal tissue grafts were also evaluated as a control. Grafted tissue was extirpated after 7, 14, 21, and 45 days. In the xenogeneic group, proliferative cell nuclear antigen-positive cells were observed 7 days after surgery, which differentiated into chondroblasts with bone morphogenetic protein-2 expression and finally formed cartilage by 14 days. Endochondral ossification was observed at 21 days, and bone replacement was completed by 45 days. No osteogenic cell activity was observed in the two other groups. Xenogeneic young periosteum thus maintained its osteogenic/chondrogenic potentiality in older rats.     

Extracorporeal shock wave‐induced proliferation of periosteal cells

The cambium cells of the periosteum are an important cell source for select tissue engineering/regenerative medicine applications due to their osteogenic and chondrogenic potential. However, the cambium layer is only 2–5 cells thick, which complicates its harvest, and the low cell number limits its suitability for certain applications. Extracorporeal shock waves (ESWs) have been reported to cause periosteal osteogenesis following cambium layer thickening. This study quantified the proliferation of cambium cells in the femur and tibia of adult rats following ESW treatment at two different energy flux densities. Four days after application of ESWs, there was a significant (3‐ to 6‐fold) increase in cambium layer thickness and cell number. Proliferation was seen with an energy flux density as low as 0.15 mJ/mm². The tibial cambium cells were more proliferative than those of the femur, with the cells closest to the ESW source proliferating the most. Within the thickened periosteum, α‐smooth muscle actin and von Willebrand Factor expression were upregulated, suggesting a vascular role in ESW osteogenesis. Bone formation was seen within the stimulated periosteum at day 4. We propose that non‐invasive ESWs can be used to rapidly stimulate cambium cell proliferation, providing a larger cell population for use as a progenitor cell source for tissue engineering applications, than can normally be provided by periosteum. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1536–1543, 2011     

富血小板纤维蛋白对人脂肪干细胞增殖和成脂分化的影响

目的 探讨自体富血小板纤维蛋白(platelet-rich fibrin,PRF)对体外培养人脂肪来源干细胞(adipose-derived stem cells,ADSCs)增殖及成脂分化的影响.方法 将自愿捐献由脂肪抽吸术获取的脂肪组织进行分离培养ADSCs并鉴定.将第3代ADSCs分为空白对照组和1个PRF膜片组(1PRFM组)和2个PRF膜片组(2PRFM组).倒置显微镜观察细胞生长情况,培养后1、2、3、4、5、6、7d采用四甲基偶氮噻唑蓝比色法(MTT)法检测细胞增殖活性.在第3、5、7、9、11和14天时采用油红O染色法检测细胞成脂分化情况.结果 随着PRFM剂量的增加,细胞增殖数量和成脂率增加,3组差异具有显著统计学意义.结论 PRF能明显促进ADSCs增殖和成脂分化,可以作为自体材料应用于脂肪组织工程的研究.     

晚期糖化终末产物对大鼠成骨细胞增殖和分化的影响

目的　通过探讨晚期糖化终末产物（ Ａｄｖａｎｃｅｄ ｇｌｙｃｏｓｙｌａｔｉｏｎ ｅｎｄｐｒｏｄｕｃｔｓ Ａ Ｇ Ｅｓ）对大鼠成骨细胞增殖和分化的影响,以期揭示老年性骨质疏松症的发病机理。方法　体外合成 Ａ Ｇ Ｅｓ 以不同浓度加入成骨细胞培养体系,作用不同时间后,用 Ｍ Ｔ Ｔ 法测定细胞增殖活性,通过测定碱性磷酸酶观察成骨细胞的分化。结果　较低剂量 Ａ Ｇ Ｅｓ 在不同的培养时间均显著促进大鼠成骨细胞增殖。较高剂量（８００ μｇ／ｍ ｌ～１ ０００ μｇ／ｍ ｌ）只有在培养２４小时具有促增殖作用。低剂量 Ａ Ｇ Ｅｓ 促进成骨细胞分化,中等剂量促分化作用延迟,高剂量无促分化活性。结论　过多的 Ａ Ｇ Ｅｓ 相对降低成骨细胞的数量和活性,使骨形成作用小于骨吸收作用,导致骨质疏松     

The relationship between periosteal division and compression or distraction of the growth plate. An experimental study in the rabbit

We subjected the proximal tibial growth plates of six-week-old rabbits to either compression or distraction of 1 kg on both legs. On one side the proximal tibial periosteum was divided circumferentially and stripped for 1 cm. After six weeks, growth was measured at both proximal and distal growth plates. Compression inhibited total tibial growth and distraction enhanced it. The compressed growth plate grew less and the distracted growth plate grew more, but there was a reciprocal change at the other end of the bone. Periosteal division enhanced growth at the adjacent growth plate but inhibited it distally; the effect of distraction was enhanced and that of compression reduced. We found reciprocal growth rates at the proximal and distal growth plates. Relatively small amounts of compression or distraction did affect total bone growth. Periosteal division appeared to induce overgrowth at least partly by a mechanical effect; it may be useful as an adjunct to other methods of leg lengthening, though not to epiphyseolysis.     

麝香配伍乳香对前列腺干细胞增殖分化作用的实验研究

目的:研究麝香、乳香配伍处理对前列腺干细胞增殖分化的影响。方法:选用7~10天龄的C57BL/6雄性幼鼠和16~18天龄的孕鼠,分别分离培养出前列腺上皮细胞和泌尿生殖窦间质(UGSM)细胞,并将二者混悬液移植到SCIDCB.17雄性小鼠的肾包膜下,30 d后收获移植物。将SCIDCB.17小鼠随机分为4组:麝香组、乳香组、麝香+乳香组、空白对照组,每组8只,按组别分别予麝香、乳香、麝香+乳香、生理盐水连续灌胃14 d后,取肾脏组织。通过HE染色形态学观察麝香配伍乳香对前列腺干细胞移植后腺管分化形成、各亚群干细胞分化状态影响,免疫组化和Western印迹检测前列腺干细胞抗原标志物P63、CD133、CD117、sca-1的表达分布情况。结果:建立了前列腺干细胞Lin-Sca-1~+CD49f~+(LSC)细胞及小鼠胚胎UGSM细胞的分离、混合培养体系;免疫组化结果示类前列腺腺泡上皮样结构P63、CD133、Sca-1、CD117染色呈阳性,证实移植物中可见前列腺腺泡上皮结构,且乳香组、麝香组的表达较空白对照组均有不同程度的增高,其中麝香+乳香组CD133、Sca-1、CD117免疫组化的积分光密度(IOD)值均显著高于单用乳香组和空白对照组(均<0.05),同时Sca-1的表达水平也显著高于单用麝香组(P<0.05),而P63的表达水平4组间无显著差异。Western印迹结果也显示,麝香+乳香组CD133、Sca-1、CD117的相对表达量均显著高于单用乳香组和空白对照组(P<0.05或<0.01),Sca-1的表达水平也显著高于单用麝香组(P<0.01)。结论:麝香、乳香配伍使用能促进前列腺干细胞的增殖再生。     

山茱萸新苷Ⅰ对成骨细胞的增殖及成骨分化的影响

目的基于Wnt信号通路及内质网应激研究山茱萸新苷Ⅰ对大鼠原代成骨细胞的增殖及成骨分化的影响。方法提取原代成骨细胞,CKK8实验检测在药物浓度下山茱萸新苷Ⅰ对成骨细胞增殖的影响;碱性磷酸酶(alkaline phosphatase,ALP)染色检测不同浓度山茱萸新苷Ⅰ干预下成骨细胞ALP的活性情况; Real-time PCR检测Wnt2、β-catenin、BMP2、OPG、NOX4、PERK基因mRNA的表达; Western blot检测Wnt2、β-catenin、PDI、CHOP和BIP蛋白表达量。结果 CKK8实验及ALP染色表明山茱萸新苷Ⅰ对成骨细胞增殖及分化有一定影响,不同浓度山茱萸新苷Ⅰ干预下成骨细胞出现不同程度的增殖;与空白对照组相比,山茱萸新苷Ⅰ组的Wnt2、BMP2、β-catenin、OPG、NOX4的mRNA表达水平显著升高(P0. 01),而PERK明显降低(P0. 01)。与空白对照组相比,山茱萸新苷Ⅰ组的成骨细胞Wnt2、β-catenin、PDI蛋白表达量显著升高(P0. 01),CHOP表达亦有轻微上升,但不明显(P0. 05),而BIP的蛋白表达水平明显降低(P0. 01)。结论山茱萸新苷Ⅰ浓度为1 mmol/mL时在促进成骨细胞的增殖分化的作用上表现最佳,其能显著升高成骨细胞Wnt2、BMP2、β-catenin、OPG和NOX4的mRNA表达水平,而降低PERK的mRNA表达水平,升高Wnt2、β-catenin、PDI的蛋白表达量,降低BIP的表达量,在山茱萸新苷Ⅰ的干预下,Wnt信号通路及内质网应激途径对成骨活动的发生发展起着重要作用。     

Effect of dialyser membrane pore size on plasma homocysteine levels in haemodialysis patients.

BACKGROUND: Hyperhomocysteinaemia is a putative risk factor for atherothrombotic cardiovascular disease in the haemodialysis population. High-dose vitamin B therapy does not entirely normalize elevated plasma total homocysteine (tHcy) levels in haemodialysis patients. Alternative therapies to reduce tHcy further are therefore required. Modifications of the dialysis regimen may result in a better removal of Hcy. We examined the effect of dialyser membrane pore size on tHcy levels in vitamin-replete chronic haemodialysis patients. METHODS: Forty-five haemodialysis patients were dialysed during 4 weeks with a low-flux, a high-flux and a super-flux membrane, in random order. Pre-dialysis tHcy was determined at baseline and every 4 weeks. In 18 patients, plasma tHcy before and after dialysis and dialysate tHcy concentrations were measured. RESULTS: Pre-dialysis tHcy decreased significantly during 4 weeks super-flux dialysis (-14.6 +/- 2.8%), whereas it remained stable during high-flux (+0.5 +/- 2.4%) and low-flux dialysis (+1.7 +/- 3.2%). The homocysteine reduction ratio was not different for the three membranes: 0.39 +/- 0.03 for the super-flux, 0.47 +/- 0.02 for the high-flux and 0.39 +/- 0.02 for the low-flux dialyser. The amount of Hcy recovered in the dialysate during a single dialysis session was also similar: 117.5 +/- 3.6 micro mol during super-flux, 95.3 +/- 11.5 micro mol during high-flux and 116.5 +/- 11.6 micro mol during low-flux dialysis. CONCLUSION: Super-flux dialysis significantly lowers tHcy in chronic haemodialysis patients. Improved removal of middle-molecule uraemic toxins with inhibitory effects on Hcy-metabolizing enzymes, rather than better dialytic clearance of Hcy itself, may explain the beneficial effect of the super-flux membrane.     

臂丛损伤神经干细胞脊髓前角移植后分化情况及对运动神经元的保护作用

目的观察臂丛根性撕脱伤后将脊髓源性神经干细胞(neuralstemcell,NSC)移植于脊髓前角后的存活、分化情况及对脊髓前角受损运动神经元的保护作用。方法取新生鼠脊髓,分离获得脊髓源性神经干细胞,体外培养、扩增、鉴定、5溴2-脱氧尿苷(BrdU)标记。取SD大鼠60只,随机分成实验组、对照组和单纯组。从后路制备C5~C7臂丛神经根性撕脱伤动物模型。实验组移植神经干细胞于C6脊髓前角,对照组移植灭活神经干细胞,单纯组不作移植。术后1、2、4、8、12周取脊髓标本进行组织学与免疫组化染色观察。结果神经干细胞移植入脊髓后能存活、分化;臂丛根性撕脱伤后脊髓前角运动神经元数目明显减少;实验组神经干细胞移植后2、4、8、12周各个时间点运动神经元的存活率均高于对照组和单纯组。结论臂丛根性撕脱伤脊髓前角神经干细胞移植后能存活并分化为神经元及星型胶质细胞,脊髓源性神经干细胞移植能明显减少前角运动神经元的继发性死亡,对脊髓前角受损运动神经元有保护作用。