骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复节段性骨缺损

目的 用自体骨髓间充质干细胞(MSCs)、骨形态发生蛋白(BMP)和纤维蛋白的复合物修复骨缺损。方法 抽取兔自体骨髓并分离和大量培养MSCs。在12只MSCs供体兔的两侧桡骨中段造成1.5cm缺损，一侧植入自体MSCs、15mgBMP和纤维蛋白的复合物(B M F)，另一侧植入自体MSCs与纤维蛋白的复合物(M F)。另一组等数量实验兔造成相同缺损后，一侧植入15mgBMP与纤维蛋白的复合物(B M)，另一侧留作空白对照。术后第2，4，8周做放射学、组织学和ECT检查。结果 B M F组在术后2周即产生填满缺损区的骨痂影，8周时骨缺损得到良好修复。B M组所产生骨痂量和修复效果均不如B M F组，但优于M F组。结论 用自体骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复骨缺损是一种切实可行和有效的方法。     

骨髓间充质干细胞修复兔骨缺损的实验研究

大段骨缺损的修复一直是骨科的热点和难点,我们通过兔桡骨缺损模型,探讨自体骨髓间充质干细胞(MSCs)修复大段骨缺损的能力。一、材料与方法1.MSCs的分离、培养:选成年新西兰大白兔15只,每只兔抽取骨髓约4ml,离心、洗涤后,细胞成分以含10 ?S的DMEM培养,不贴壁的细胞经换液除去,贴壁细胞经增殖、传代,MSCs逐步纯化。2 .细胞—载体复合物的准备:另取兔松质骨,制成12mm×4mm×4mm的冻干松质骨块,作为载体。取第3代MSCs ,以无血清DMEM制成5×10 6cells/ml的细胞悬液,通过轻度负压使细胞随培养液渗入骨块内部,制成细胞—载体复合物备…     

骨形态发生蛋白2基因治疗与缓释载体修复骨缺损的比较研究

[目的]比较骨形态发生蛋白2（BMP-2）基因治疗与生长因子缓释方法修复节段性骨缺损效果。[方法]于兔双侧桡骨中段造成1．5cm骨缺损，采用4种方法修复：A组植入转基因骨髓间质干细胞（MSCs）与PLA／PCL（聚乳酸／聚己内酯）支架的复合物；B组植入单纯MSCs与含重组BMP-2的PLA／PCL缓释载体的复合物；C组植入单纯MSCs与PLA／PCL复合物；D组植入单纯PLA／PCL。术后4、8、12周行X线、组织学、生物力学和骨密度等检测，[结果]A组体内植入4周后，成骨细胞和间质细胞呈BMP-2强阳性表达；其成骨速度及成骨质量均明显优于B组，12周时骨缺损完全修复、C组成骨能力较弱，而D组则无新骨形成，残留骨缺损。[结论]BMP-2基因治疗是修复节段性骨缺损的好方法。     

骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复节段性骨缺损

目的　用自体骨髓间充质干细胞 (MSCs)、骨形态发生蛋白 (BMP)和纤维蛋白的复合物修复骨缺损。方法　抽取兔自体骨髓并分离和大量培养MSCs。在 12只MSCs供体兔的两侧桡骨中段造成 1 5cm缺损 ,一侧植入自体MSCs、 15mgBMP和纤维蛋白的复合物 (B +M +F) ,另一侧植入自体MSCs与纤维蛋白的复合物 (M +F)。另一组等数量实验兔造成相同缺损后 ,一侧植入 15mgBMP与纤维蛋白的复合物 (B +M ) ,另一侧留作空白对照。术后第 2 ,4 ,8周做放射学、组织学和ECT检查。结果　B +M +F组在术后 2周即产生填满缺损区的骨痂影 ,8周时骨缺损得到良好修复。B +M组所产生骨痂量和修复效果均不如B +M +F组 ,但优于M +F组。结论　用自体骨髓间充质干细胞复合骨形态发生蛋白和纤维蛋白修复骨缺损是一种切实可行和有效的方法。     

涂层双相陶瓷复合BMSC修复骨缺损的实验观察

目的比较双相陶瓷（Biphasie calcium phosphate，BCP）经低结晶羟基磷灰石（Low crystalline hydroxyapatite，LcHA）涂覆改性后构建的组织工程化骨（LcBCP）与单纯BCP复合骨髓基质干细胞（Bone marrow stromal cells，BMSCs）修复兔桡骨节段性缺损的成骨差异。方法BMSCs复合LcBCP（实验组）修复12只兔左侧桡骨15mm缺损；BMSCs复合BCP（对照组）植入右侧桡骨同样大小缺损，植入后第4、8和12周取材，通过大体形态、组织学、影像学和生物力学检测骨缺损修复效果。结果BMSCs—LcBCP复合物在体内骨缺损处生长良好。X线检测显示实验组连接处骨痂形成，对照组连接处在各个时间点愈合稍差。12周时，实验组骨修复良好，髓腔再通，组织学显示板层骨形成，连接处骨性愈合；对照组连接处尚有较多编织骨形成。实验组和对照组生物力学检测有统计学差异。结论BMSCs—LcBCP复合物可修复兔桡骨节段性缺损，低品态羟基磷灰石涂层有助于增强双相陶瓷的成骨能力。     

基因修饰的组织工程骨修复节段性骨缺损及相关免疫学研究

目的：观察骨形态发生蛋白-2（BMP-2）基因修饰的组织工程骨修复节段性骨缺损效果及异种骨支架体内应用的安全性。方法：①制备去抗原牛松质骨块（BCB），植入小鼠股四头肌袋内，术后行淋巴细胞转化试验和组织学观察。②在腺病毒载体介导下将BMP-2基因导入兔骨髓间质干细胞后，种植到BCB支架中，构建基因修饰的组织工程骨。于兔双侧桡骨中段造成15mm骨缺损，采用5种方法进行处理：BMP-2基因转染细胞＋B（B（A组）；未转染细胞＋重组BMP-2＋BCB（B组）；对照基因转染细胞＋BCB（C组）；未转染细胞＋B（B（D组）；单纯BCB（E组）。术后4、8、12周行X线、组织学和生物力学检测。结果：①BCB具有较低的抗原性和良好的组织相容性；②A组术后4周诱导生成软骨组织并向编织骨转化，12周骨缺损修复，髓腔再通，新骨强度明显优于其他各组（P〈0．01）。结论：BMP-2基因修饰的组织工程骨是修复节段性骨缺损的好方法。     

骨形态发生蛋白2基因治疗在修复骨缺损中对血管化影响的实验研究

目的观察骨形态发生蛋白-2（BMP-2）基因治疗在修复骨缺损中对血管化的影响。方法分离培养兔骨髓基质干细胞（MSCs），经BMP-2腺病毒载体转染后复合异种骨支架移植修复1．5cm桡骨缺损。分为5组：①BMP-2基因转染细胞＋去抗原牛松质骨支架（BCB）；②未转染细胞＋重组BMP-2＋BCB；③对照基因转染细胞＋BCB；④未转染细胞＋BCB；⑤BCB。术后4、8、12周行微血管墨汁灌注、血管内皮生长因子（VEGF）免疫组化染色、组织学等检测。结果术后4周，基因治疗组骨间血管的密度在周边区域较高，通常每一个骨小梁孔隙中都有一支新形成的小血管；透射电镜见成骨细胞总是毗邻血管内皮细胞而存在，并随着微血管的增生而逐渐向骨细胞发展；间质细胞VEGF表达明显增强。结论BMP-2基因治疗可通过上调VEGF表达，间接诱导移植骨血管化，对骨不连、骨缺损的治疗具有重要意义。     

异位成骨材料修复新西兰兔桡骨干骨缺损

目的探索异位成骨材料修复新西兰兔桡骨干骨缺损的可行性。方法13只新西兰白兔(4周龄,体质量1.5 kg)作为实验动物。取新西兰白兔骨髓间充质干细胞进行成骨诱导,2周后予碱性磷酸酶染色确认其成骨活性。取诱导后的间充质干细胞悬液与β-磷酸三钙生物陶瓷共培养,3天后植入4只新西兰白兔股骨骨膜旁。于植入后第8周收取异位成骨材料,行骨组织切片检查。取8只新西兰兔,制作桡骨干骨缺损动物模型,并将其均分为实验组(4只)和对照组(4只),分别植入异位成骨材料和β-磷酸三钙生物陶瓷修复骨缺损,于术后4、8、12周行手术侧X线摄片,术后12周取修复处骨,行组织切片检查。结果异位成骨植入第8周见植入物成骨完整,获取的成骨材料组织切片显示与股骨干骨细胞结构相似。骨缺损修复动物实验术后X线摄片显示,实验组修复效果显著优于对照组;术后12周修复处骨组织切片显示,实验组新生骨与周边骨组织边界消失,融合度好,骨小梁密度高,新生血管较多。结论异位成骨材料可用于兔桡骨干骨缺损修复。     

组织工程骨膜及脱蛋白骨修复兔桡骨大段骨缺损的早期观察

[目的]比较组织工程骨膜及脱蛋白骨对兔桡骨大段骨缺损的早期修复效果,研究组织工程骨膜移植修复大段骨缺损的可行性。[方法]分离兔骨髓间充质干细胞体外诱导增殖,制成细胞悬液接种于猪小肠黏膜下层表面,构建组织工程骨膜。3个月龄新西兰大白兔24只,随机分为3组各8只,手术制备单侧桡骨干30 mm骨膜-骨缺损模型,A组缺损区植入组织工程骨膜骨修复,B、C组则分别以猪小肠黏膜下层和脱蛋白骨作为对比。术后4周,行X线检查并杀取标本予以组织学染色及评分。[结果]经放射学和组织学观察,在A组中缺损区可见大量新骨形成并桥连两缺损端;而在B组和C组中无明显新骨产生。[结论]使用组织工程骨膜修复同种异体兔桡骨大段骨缺损是可行的,且明显优于脱蛋白骨。     

PRP/脱蛋白异种骨修复兔桡骨骨缺损的组织学研究

目的探讨富血小板血浆(platelet-rich plasma,PRP)和脱蛋白牛松质骨复合物修复节段性骨缺损的疗效。方法新西兰大白兔28只,切除兔桡骨中下段1 cm的骨质,其中空白组骨缺损区不作处理,试验组骨缺损区植入PRP 脱蛋白牛松质骨,对照组骨缺损区单纯植入脱蛋白牛松质骨。于术后第2、4、8、12周分别处死7只试验动物,结果按Lane骨移植组织学评分标准评分,并进行图像分析,测其新生骨面积。结果术后实验组Lane骨移植组织学评分高于对照组,实验组新生骨面积明显高于对照组。结论复PRP异种脱蛋白骨可用于节段性骨缺损的修复,在骨缺损的修复中具有促进作用成骨方式为骨传导和骨诱导。     

Collagen‐containing scaffolds enhance attachment and proliferation of non‐cultured bone marrow multipotential stromal cells

Large bone defects are ideally treated with autografts, which have many limitations. Therefore, osteoconductive scaffolds loaded with autologous bone marrow (BM) aspirate are increasingly used as alternatives. The purpose of this study was to compare the growth of multipotential stromal cells (MSCs) from unprocessed BM on a collagen‐containing bovine bone scaffold (Orthoss^® Collagen) with a non‐collagen‐containing bovine bone scaffold, Orthoss^®. Another collagen‐containing synthetic scaffold, Vitoss^® was included in the comparison. Colonization of scaffolds by BM MSCs (n = 23 donors) was evaluated using microscopy, colony forming unit‐fibroblast assay and flow‐cytometry. The number of BM MSCs initially attached to Orthoss^® Collagen and Vitoss^® was similar but greater than Orthoss^® (p = 0.001 and p = 0.041, respectively). Furthermore, the number of MSCs released from Orthoss^® Collagen and Vitoss^® after 2‐week culture was also higher compared to Orthoss^® (p = 0.010 and p = 0.023, respectively). Interestingly, collagen‐containing scaffolds accommodated larger numbers of lymphocytic and myelomonocytic cells. Additionally, the proliferation of culture‐expanded MSCs on Orthoss^® collagen and Vitoss^® was greater compared to Orthoss^® (p = 0.047 and p = 0.004, respectively). Collectively, collagen‐containing scaffolds were superior in supporting the attachment and proliferation of MSCs when they were loaded with unprocessed BM aspirates. This highlights the benefit of collagen incorporation into bone scaffolds for use with autologous bone marrow aspirates as autograft substitutes. © 2015 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:597–606, 2016.     

Glucocorticoid decreases circulating osteoprotegerin (OPG): possible mechanism for glucocorticoid induced osteoporosis.

BACKGROUND: Osteoporosis is a well known side-effect of long-term treatment with glucocorticoids. However, the precise mechanism of this disorder is unclear. Recently, osteoprotegerin (OPG) [osteoclastogenesis inhibitory factor (OCIF)] has been identified as a novel cytokine, which inhibits differentiation and activation of osteoclast. In the present study, in order to clarify the roles of OPG in the development of glucocorticoid-induced osteoporosis, we measured circulating OPG before and after glucocorticoid therapy. METHODS: The study subjects were 12 patients (five males, seven females, 53.4 +/- 4.8 [SE] years) with various renal diseases that required glucocorticoid therapy. All patients received glucocorticoids for the first time. Treatment was initiated at an average dose of 32.5 +/- 3.0 mg per day. Serum OPG was measured using enzyme-linked immunosorbent assay (ELISA). Laboratory data, markers of bone metabolism and circulating OPG were compared before and after treatment for 4 weeks. RESULTS: Serum OPG prior to glucocorticoid therapy was positively and independently correlated with serum creatinine. Serum OPG decreased significantly (P: < 0.0001) from 1.03 +/- 0.14 to 0.77 +/- 0.12 ng/ml after short-term administration of glucocorticoids. Furthermore, serum osteocalcin as a marker of bone formation was also reduced markedly (P: = 0.001). On the other hand, there were no remarkable changes in serum calcium, total alkaline phosphatases, creatinine and intact parathyroid hormone in response to glucocorticoid administration. CONCLUSION: These findings indicate that short-term administration of glucocorticoids significantly suppresses serum OPG and osteocalcin. It might participate in the development of glucocorticoid-induced osteoporosis via an enhancement of bone resorption and suppression of bone formation.     

生物活性骨水泥的孔隙率与骨水泥全髋关节置换术后假体早期位移的相关性研究

[目的]比较全髋关节置换术后早期新型生物活性骨水泥即含锶羟基磷灰石骨水泥(Sr-HA)与传统的聚甲基丙烯酸甲酯骨水泥(PMMA)对假体固定的效果.[方法]从2008年5月～2009年12月在香港玛丽医院行全髋关节置换术的9例(10膝)患者随机分为观察组和对照组,分别使用Sr-HA骨水泥与PMMA骨水泥同定股骨柄,于术后1周、3个月、6个月及12个月随访使用放射立体照相测量分析技术(RSA)测量股骨柄相对于股骨的位移,记录Harris髋关节评分,并于实验室使用显微CT扫描测鼍两种骨水泥的孔隙率进行比较.[结果]所有患者手术经过顺利,无血管神经损伤、肺栓塞等并发症.RSA结果显示Sr-HA组有3例假体的下沉均超过1 mm,而PMMA组假体的下沉全部在1 mm以内.两组患者的Harris髋关节评分在术后均有明显改善.显微CT扫描结果显示Sr-HA骨水泥的孔隙率高于PMMA骨水泥,两者之间差异有统计学意义(P＜0.01).[结论]两组患者术后均无明显临床症状,含锶羟基磷灰石骨水泥相对较高的孔隙率可能是导致全髋关节置换术后早期假体位移的原因.     

Remarkable increase in lumbar spine bone mineral density and amelioration in biochemical markers of bone turnover after parathyroidectomy in elderly patients with primary hyperparathyroidism: a 5-year follow-up study

We evaluated the efficacy of parathyroidectomy (PTX) on bone mineral density (BMD) and hormonal and biochemical markers of bone metabolism in elderly primary hyperparathyroidism (PHPT) patients, and followed these patients for 5 years after PTX. Eleven PHPT patients were enrolled and were followed for 5 years by measuring lumbar spine BMD (LSBMD), femoral BMD (FBMD), radial BMD (RBMD), parathyroid hormone (PTH), 1,25-dihydroxyvitamin D [1,25(OH)₂D], serum calcium (SCa), inorganic phosphate (iP), bone-specific alkaline phosphatase (BAP), intact osteocalcin (IOC), urinary excretion of type I collagen cross-linked N-telopeptide (NTx), and urinary deoxypyridinoline (DPD). PTX produced significant increases in LSBMD of 12%, 19%, and 29% as compared with pretreatment levels after 1, 3, and 5 years, respectively (P < 0.01, compared to baseline), whereas there was no significant increase in FBMD and a slight decrease in RBMD. SCa and iP levels remained normal over the five years. PTX also resulted in significant decreases in PTH, 1,25(OH)₂D, BAP, IOC, NTx, and DPD that continued for at least 3 years after PTX. In conclusion, PTX seemed effective to normalize various markers of bone metabolism in elderly PHPT patients and is recommended to patients with low LSBMD to prevent future fractures. On the other hand, the use of PTX for low FBMD or RBMD patients requires further discussion.     

Clinical Efficacy Analysis of Percutaneous Kyphoplasty Combined with Zoledronic Acid in the Treatment and Prevention of Osteoporotic Vertebral Compression Fractures

ABSTRACT

Objective: The aim of this study is to investigate the clinical efficacy of percutaneous kyphoplasty (PKP) combined with zoledronic acid (aclasta) in the treatment and prevention of osteoporotic vertebral compression fractures (OVCF). Method: A total of 104 patients with OVCF were treated with PKP from February 15, 2014 to January 17, 2016. All patients were randomly assigned to control and experimental groups, each group having 52 patients. All patients in the control group were treated with PKP. On the other hand, all patients in the experimental group were treated with PKP combined with aclasta. To evaluate the clinical efficacy, Visual Analogue Scale (VAS), Oswestry disability index (ODI), bone mineral density (BMD), and bone metabolism (N-MID and β-CTX) were evaluated during the follow-up period. Result: One week after operation, the value of VAS and ODI improved in both groups. One year after operation, these worsened rapidly in the control group but not in the experimental group. Six months and one year after operation, the BMD improved significantly but the bone metabolism decreased significantly in the experimental group. In contrast, the BMD and bone metabolism did not change in the control group. Moreover, four patients again suffered from OVCF in the control group, and three patients had fever symptoms in the experimental group during the follow-up period. Conclusions: Results of this study indicate that the clinical efficacy of PKP combined with aclasta in the treatment and prevention of OVCF is significant.     

隔膜引导下血管内皮生长因子复合煅烧骨对骨缺损修复作用的实验研究

[目的]研究隔膜引导下VEGF复合煅烧骨（TBC）修复骨缺损的作用，探讨其临床应用前景。[方法]新西兰大白兔80只，建立桡骨干中段10mm骨缺损模型。A组植入e—PTFE膜包绕的VEGF复合TBC，B组植入VEGF复合的TBC，C组植入e-PTFE膜包绕的TBC，D组单纯植入TBC。术后第2、4、6、8和12周行X线、组织学观察，测定骨密度并分析其成骨量。[结果]D组的骨缺损修复效果最差，B、C组中等，A组最佳。A组在各时段上其骨密度测量的结果优于B、C、D组，差异性显著（P〈0．01）。[结论]e—PTFE膜包绕的VEGF复合TBC能够更好地修复骨缺损，是一种较为理想的骨组织工程材料，具有良好的临床应用前景。     

High Degree of Discordance Between Three-Dimensional and Two-Dimensional Lumbar Spine Bone Mineral Density in Turner''s Syndrome

Low bone mineral density (BMD) measured by dual-energy X-ray absorptiometry (DXA) has been described in Turner's syndrome (TS). One of the error factors of DXA is short stature, a common finding in TS patients. Aimed to evaluate the influence of a low stature on BMD, we compared the two-dimensional (2D) or conventional BMD (cBMD) with three-dimensional (3D) or volumetric BMD (vBMD) in 62 females (10 to 48 yr old) with TS diagnosis in a case control study. They were compared to 102 normal females (7 to 45 yr old) grouped by age-ranges. All patients were subjected to a lumbar spine densitometry by DXA in the PA and lateral projections, obtained the cBMD and vBMD and calculated for the apparent BMD (appBMD). In TS, the mean of Z-score for cBMD was significantly lower than that for vBMD and for appBMD (-2.31 +/- 1.42; -0.64 +/- 1.55; and -1.72 +/- 1.5; respectively). Most of the patients (83.8%) had a Z-score <-1 for cBMD, whereas the majority (58.1%) had a Z-score <-1 for vBMD. Concluding, the cBMD underestimates the bone mass of the lumbar spine in patients with TS inducing to false diagnoses of bone fragility. Volumetric BMD approached the bone mass of control patients, while appBMD just partially do that.     

EXPERIMENTAL CALVARIAL GROWTH DISTURBANCE BY MICRO-PLATE AND SCREW FIXATION

A study was designed to help ascertain the effects of rigid internal fixation in the growing skull. Five piglets underwent plating of the left coronal suture at 3 weeks of age. At the time of surgery metal markers were placed to follow bone growth. Cephalometric radiographs, direct osteometry at sacrifice, gross pathological examination and specimen radiography revealed a localized disturbance of growth. There was a restriction of sutural displacement and appositional bone growth at the site of the plate. Local restriction of sutural growth was almost complete during the experimental period. There was an alteration to a ‘mature’ suture morphology, without synostosis, of the suture on the plated side not seen on the control side. A plagiocephalic pig was not produced nor was there an orbital deformity. A large local contour deformity developed and by the end of the experimental period the plate had become completely incorporated in bone. These effects were statistically significant as well as clinically significant. The clinical use of this type of fixation in reconstructive surgery of the infant craniofacial skeleton is questioned in light of the result.     

Gene-mediated osteogenic differentiation of stem cells by bone morphogenetic proteins-2 or -6.

Bone marrow-derived mesenchymal stem cells (BMDMSC) hold promise for targeted osteogenic differentiation and can be augmented by delivery of genes encoding bone morphogenetic proteins (BMP). The feasibility of promoting osteogenic differentiation of BMDMSC was investigated using two BMP genes in monolayer and three-dimensional alginate culture systems. Cultured BMDMSC were transduced with E1-deleted adenoviral vectors containing either human BMP2 or BMP6 coding sequence under cytomegalovirus (CMV) promoter control [17:1 multiplicities of infection (moi)] and either sustained in monolayer or suspended in 1 mL 1.2% alginate beads for 22 days. Adenovirus (Ad)-BMP-2 and Ad-BMP-6 transduction resulted in abundant BMP-2 and BMP-6 mRNA and protein expression in monolayer culture and BMP-2 protein expression in alginate cultures. Ad-BMP-2 and Ad-BMP-6 transduced BMDMSC in monolayer had earlier and robust alkaline phosphatase-positive staining and mineralization and were sustained for a longer duration with better morphology scores than untransduced or Ad-beta-galactosidase-transduced cells. Ad-BMP-2- and, to a lesser degree, Ad-BMP-6-transduced BMDMSC suspended in alginate demonstrated greater mineralization than untransduced cells. Gene expression studies at day 2 confirmed an inflammatory response to the gene delivery process with upregulation of interleukin 8 and CXCL2. Upregulation of genes consistent with response to BMP exposure and osteogenic differentiation, specifically endochondral ossification and extracellular matrix proteins, occurred in BMP-transduced cells. These data support that transduction of BMDMSC with Ad-BMP-2 or Ad-BMP-6 can accelerate osteogenic differentiation and mineralization of stem cells in culture, including in three-dimensional culture. BMP-2-transduced stem cells suspended in alginate culture may be a practical carrier system to support bone formation in vivo. BMP-6 induced a less robust cellular response than BMP-2, particularly in alginate culture.