胶原杂化去抗原松质骨载体复合自体间充质干细胞修复骨软骨缺损

目的制备胶原杂化去抗原松质骨载体并复合自体间充质干细胞修复关节骨软骨缺损。方法脱脂、脱蛋白和脱钙的方法来制备去抗原松质骨载体，酸解-酶解法制备胶原进而杂化松质骨载体。扩增间充质干细胞，接种到胶原杂化松质骨载体，植入左膝关节（实验侧），右膝关节植入没有复合细胞的胶原杂化去抗原松质骨载体（对照侧），在第6、12、24、48周时结合Pineda评分观察实验侧和对照侧的骨软骨缺损的组织修复情况。结果实验侧自12周时出现初步的软骨表型，24周时最为明显，48周有部分退变；软骨下骨部分逐渐恢复。对照侧软骨缺损一直以纤维组织充填，软骨下骨有部分恢复。上述各时间点实验侧和对照侧的Pineda评分为16．600±0．966。17．600±0．699（6周）；6．600±0．843、14．800±0．789（12周）；3．300±0．483、13．600±0．843（24周）；3．500±0．527、14．100±0．876（48周），差异均有统计学意义（P〈0．05）。结论胶原杂化的去抗原松质骨载体复合自体间充质干细胞能够修复关节的骨软骨缺损。     

Autologous osteochondral grafting for talar cartilage defects

The purpose of this study was to evaluate the clinical results of Osteochondral Autograft Transfer System (OATS) for the treatment of symptomatic osteochondral defects of the talus using standardized outcome analysis. Nineteen patients with symptomatic osteochondral defect (OCD) of the talus were treated with autologous osteochondral grafting. There were six men and 13 women. The average age was 32 years (range, 18 to 48 years). The average duration of symptoms prior to surgery was 4.2 years (range, three months to 12 years). All patients had failed nonoperative treatment, and 13 (68%) patients had failed prior excision, curettage and/or drilling of the lesion. The average size of the lesion prior to autografting was 12 mm x 10 mm (range, 10 x 5 mm to 20 x 20 mm). Donor plugs were harvested from the trochlear border of the ipsilateral femoral condyle. Ankle exposure was obtained with a medial malleolar osteotomy in 13 patients, arthrotomy in five patients and lateral malleolar osteotomy in one patient. Clinical evaluations were performed for both the recipient ankle and donor knee using the AOFAS Ankle/Hindfoot Scale and Lysholm knee scale, respectively. The average follow-up time was 16 months (range, 12 to 30 months). The average postoperative AOFAS ankle score was 88 (range, 60 to 100). Most patients had occasional mild pain, but excellent function, range of motion, stability and alignment. The average postoperative ankle score for the 13 patients who failed prior surgery was 91 (range, 84 to 100). The average postoperative Lysholm knee score was 97 (range, 87 to 100). Only two patients had mild knee pain. Postoperative radiographs were available for 13 patients. There was no evidence of graft subsidence and all grafts healed. All malleolar osteotomies united. Seventeen (89%) patients said that they would undergo the procedure again. The results of osteochondral autograft transplant for OCD lesions of the talus demonstrate excellent postoperative ankle scores including improvement of pain and function with minimal knee donor site morbidity. Also, our results indicate that this is an effective salvage procedure following failed previous procedures and for patients with longstanding symptoms.     

Autologous osteochondral graft for traumatic defects of finger joints

Finger joint defects in 16 adults were treated with an autologous osteochondral graft from the base of the second metacarpal, the radial styloid, the base of the third metacarpal or the trapezoid and these patients were followed up from between 12 and 62 months. There was no donor site morbidity. One patient had resorption of the graft and developed pain. The joint was subsequently fused. The mean range of movement was 55.8% of the opposite normal joint. At follow up, 15 patients had no discomfort or mild discomfort. Three had mild narrowing of the joint space and two had slight joint subluxation. Only two patients with concomitant severe injury to the same limb had difficulty performing daily activities. Ten were open injuries and these had poorer outcomes. A hemicondylar defect of a finger joint can be treated using an osteochondral graft obtained from the same hand.     

自体骨软骨镶嵌移植修复猪膝关节骨软骨缺损

目的: 探讨猪自体骨软骨镶嵌移植治疗股骨负重区骨软骨复合缺损的优缺点。方法: 采用德国Arthrex公司OATS软骨移植器械, 进行长枫杂交仔猪同关节非负重区骨软骨移植修复负重区骨软骨缺损。结果: 12周后发现骨软骨柱移植后存活良好, 移植软骨中心区无明显凹陷及退变, 甚至稍有凸起, 质地同正常软骨; 与周围正常软骨界限仍清晰, 交界区软骨整合差, 有明显裂隙存在; 骨软骨供区有明显凹陷, 为白色组织充填, 质软, 周围可见正常软骨退变。结论: 自体骨软骨镶嵌移植具有移植软骨固定可靠、软骨细胞存活率高等优点, 但也有交界区整合差, 为纤维软骨修复等缺点。     

The secretory profiles of cultured human articular chondrocytes and mesenchymal stem cells: implications for autologous cell transplantation strategies

This study was undertaken to compare the phenotype of human articular chondrocytes (ACs) and bone marrow-derived mesenchymal stem cells (MSCs) after cell expansion by studying the spectrum of proteins secreted by cells into the culture medium. ACs and MSCs were expanded in monolayer cultures for some weeks, as done in standard cell transplantation procedures. Initially, the expression of cartilage signature genes was compared by real-time PCR. Metabolic labeling of proteins (SILAC) in combination with mass spectrometry (LC/MS-MS) was applied to investigate differences in released proteins. In addition, multiplex assays were carried out to quantify the amounts of several matrix metalloproteases (MMPs) and their natural inhibitors (TIMPs). Expanded chondrocytes showed a slightly higher expression of cartilage-specific genes than MSCs, whereas the overall spectra of released proteins were very similar for the two cell types. In qualitative terms MSCs seemed to secrete similar number of extracellular matrix proteins (43% vs. 45% of total proteins found) and catabolic agents (9% vs. 10%), and higher number of anabolic agents (12 % vs. 7%) compared to ACs. Some matrix-regulatory agents such as serpins, BMP-1, and galectins were detected only in MSC supernatants. Quantitative analyses of MMPs and TIMPs revealed significantly higher levels of MMP-1, MMP-2, MMP-3, and MMP-7 in the medium of ACs. Our data show that after the expansion phase, both ACs and MSCs express a dedifferentiated phenotype, resembling each other. ACs hold a phenotype closer to native cartilage at the gene expression level, whereas MSCs show a more anabolic profile by looking at the released proteins pattern. Our data together with the inherent capability of MSCs to maintain their differentiation potential for longer cultivation periods would favor the use of these cells for cartilage reconstruction.     

Autologous mesenchymal stem cells in chronic spinal cord injury

Spinal cord injury (SCI) occurs in the most productive part of life. Treatment options for treatment of chronic SCI are few and have limited impact on clinical outcome. Central nervous system (CNS) has limited intrinsic regeneration capability. The study included patients with chronic complete SCI. Previously harvested autologous mesenchymal stem cells were administered at the site of injury after a laminectomy. Follow-up was done by a neutral examiner not involved in the surgery every 3 months. One patient had improvement in motor power. Two patients had a patchy improvement in pin prick sensation below the level of injury. Three different, progressively increasing doses did not result in improvement in the clinical outcome. Though the administration of allogenic human mesenchymal stem cells is safe in patients with SCI, it may not be efficacious; especially in patients with chronic SCI.     

Chemotaxis of human articular chondrocytes and mesenchymal stem cells

Migration of chondrocytes and mesenchymal stem cells (MSCs) may be important in cartilage development, tissue response to injury, and in tissue engineering. This study analyzed growth factors and cytokines for their ability to induce migration of human articular chondrocytes and bone marrow‐derived mesenchymal stem cells in Boyden chamber assays.In human articular chondrocytes serum induced dose‐ and time‐dependent increases in cell migration. Among a series of growth factors and cytokines tested only PDGF induced a significant increase in cell migration. The PDGF isoforms AB and BB were more potent than AA. There was an aging‐related decline in the ability of chondrocytes to migrate in response to serum and PDGF. Human bone marrow MSC showed significant chemotaxis responses to several factors, including FBS, PDGF, VEGF, IGF‐1, IL‐8, BMP‐4, and BMP‐7. In summary, these results demonstrate that directed cell migration is inducible in human articular chondrocytes and MSC. PDGF is the most potent factor analyzed, and may be useful to promote tissue integration during cartilage repair or tissue engineering. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:1407–1412, 2008     

Use of autologous grafts for reconstruction of osteochondral defects of the knee.

This study reports on 13 patients (mean age: 31 years) with a femoral condyle defect >1.5 cm2 who underwent treatment with an osteochondral graft of the same size obtained from the superior aspect of the lateral condyle, preserving the patellar groove. Mean follow-up was 61.5 months (range: 13-141 months). Twelve results were rated clinically as satisfactory with patients able to resume their normal pre-injury level of activity, and 1 case was rated as poor. No patient reported any patellar problems. Radiographic and computed tomographic evaluation demonstrated good integration of the graft in the host bone. The results of this technique at relatively long-term follow-up are encouraging, with a high percentage of subjective satisfaction. This technique appears to be reliable and provides a valid solution for treatment of wide cartilage defects when other techniques are too complex or inadequate.     

Fate of transplanted bone-marrow-derived mesenchymal cells during osteochondral repair using transgenic rats to simulate autologous transplantation

OBJECTIVE: The fate of transplanted cells used in tissue engineering strategies should be followed. With this aim in view, the survival of transplanted bone-marrow-derived mesenchymal cells within osteochondral defects was determined using transgenic rats to simulate autologous transplantation. DESIGN: An autologous transplantation model was simulated using transgenic rats - whose transgenes produce no foreign proteins - as donors, and wild-type rats as recipients. Dense masses of mesenchymal cells were prepared from the transgenic rats using the hanging-drop culture technique. These cell masses were then transplanted into osteochondral defects created within the medial femoral condyle of wild-type rats, wherein they are affixed with fibrin glue. The course of repair was assessed histologically. The survival of the transplanted cells was ascertained by in situ hybridization of the transgenes. RESULTS: Twenty-four weeks after transplantation, the defects were repaired with hyaline-like cartilage, which was thicker than normal, and with subchondral bone. Using the in situ hybridization technique, cells derived from the transplanted ones were detected within both the cartilaginous and the subchondral bone layers. CONCLUSION: Using this simulated autologous transplantation model, the survival of transplanted mesenchymal cells was monitored in vivo. The findings indicate that the transplanted mesenchymal cells contributed to the repair of the osteochondral defects.     

Autologous chondrocyte implantation versus matrix-induced autologous chondrocyte implantation for osteochondral defects of the knee: a prospective, randomised study

Autologous chondrocyte implantation (ACI) is used widely as a treatment for symptomatic chondral and osteochondral defects of the knee. Variations of the original periosteum-cover technique include the use of porcine-derived type I/type III collagen as a cover (ACI-C) and matrix-induced autologous chondrocyte implantation (MACI) using a collagen bilayer seeded with chondrocytes. We have performed a prospective, randomised comparison of ACI-C and MACI for the treatment of symptomatic chondral defects of the knee in 91 patients, of whom 44 received ACI-C and 47 MACI grafts. Both treatments resulted in improvement of the clinical score after one year. The mean modified Cincinnati knee score increased by 17.6 in the ACI-C group and 19.6 in the MACI group (p = 0.32). Arthroscopic assessments performed after one year showed a good to excellent International Cartilage Repair Society score in 79.2% of ACI-C and 66.6% of MACI grafts. Hyaline-like cartilage or hyaline-like cartilage with fibrocartilage was found in the biopsies of 43.9% of the ACI-C and 36.4% of the MACI grafts after one year. The rate of hypertrophy of the graft was 9% (4 of 44) in the ACI-C group and 6% (3 of 47) in the MACI group. The frequency of re-operation was 9% in each group. We conclude that the clinical, arthroscopic and histological outcomes are comparable for both ACI-C and MACI. While MACI is technically attractive, further long-term studies are required before the technique is widely adopted.     

全关节镜下基质诱导的自体软骨细胞移植修复膝软骨缺损

[目的]介绍全关节镜下自体软骨细胞移植修复膝软骨缺损的技术及疗效。[方法]选取2016年8月～2018年8月,共有22例膝软骨缺损患者,初次手术取患膝非负重区软骨体外培养、扩增并与胶原纤维支架复合,3周后接受自体软骨细胞移植,全程均在关节镜下进行。术后3、6、12个月随访。[结果]所有患者手术顺利,无严重并发症发生。与术前相比较,术后12个月VAS评分显著降低[(5.36±0.95) vs(0.32±0.48),P0.001],Lysholm评分显著增加[(59.18±4.92) vs(90.64±1.84),P0.001]。术后12个月T2 Mapping核磁Z值较术初显著改善[(0.42±0.06) vs(0.89±0.08),P0.001]。[结论]全关节镜下自体软骨细胞移植修复膝关节缺损可以迅速改善症状,无严重并发症。     

Autologous transplantation of amniotic fluid-derived mesenchymal stem cells into sheep fetuses

Long-term engraftment and phenotype correction has been difficult to achieve in humans after in utero stem cell transplantation mainly because of allogeneic rejection. Autologous cells could be obtained during gestation from the amniotic fluid with minimal risk for the fetus and the mother. Using a sheep model, we explored the possibility of using amniotic fluid mesenchymal stem cells (AFMSCs) for autologous in utero stem cell/gene therapy. We collected amniotic fluid (AF) under ultrasound-guided amniocentesis in early gestation pregnant sheep (n = 9, 58 days of gestation, term = 145 days). AFMSCs were isolated and expanded in all sampled fetal sheep. Those cells were transduced using an HIV vector encoding enhanced green fluorescent protein (GFP) with 63.2% (range 38.3-96.2%) transduction efficiency rate. After expansion, transduced AFMSCs were injected into the peritoneal cavity of each donor fetal sheep at 76 days under ultrasound guidance. One ewe miscarried twin fetuses after amniocentesis. Intraperitoneal injection was successful in the remaining 7 fetal sheep giving a 78% survival for the full procedure. Tissues were sampled at postmortem examination 2 weeks later. PCR analysis detected GFP-positive cells in fetal tissues including liver, heart, placenta, membrane, umbilical cord, adrenal gland, and muscle. GFP protein was detected in these tissues by Western blotting and further confirmed by cytofluorimetric and immunofluorescence analyses. This is the first demonstration of autologous stem cell transplantation in the fetus using AFMSCs. Autologous cells derived from AF showed widespread organ migration and could offer an alternative way to ameliorate prenatal congenital disease.     

Estrogen reduces cellular aging in human mesenchymal stem cells and chondrocytes

Chondrocyte aging is associated with cartilage degeneration and senescence impairs the regenerative potential of mesenchymal stem cells (MSCs). Estrogen exerts profound effects on human physiology including articular cartilage and MSCs. The present study should analyze the effects of pre‐ and postmenopausal estrogen concentrations on chondrogenic cells. Physiologic premenopausal concentrations of 17β‐estradiol (E₂) significantly decelerated telomere attrition in MSCs and chondrocytes while postmenopausal E₂ concentration had no significant effects. The estrogen agonist–antagonist tamoxifen did not affect telomere biology, but inhibited the E₂‐stimulated reduction in telomere shortening. E₂ and tamoxifen did not influence cell proliferation, cell morphology, and β‐galactosidase staining in chondrogenic cells. E₂ treatment did not affect the telomere‐associated proteins TRF1 and TRF2. E₂ had no regulatory effects on the expression rates of the cell cycle regulator p21 and the DNA repair proteins SIRT1 and XRCC5. In spite of reducing telomere shortening in aging MSCs and chondrocytes, estrogen is not able to prevent somatic cells from replicative exhaustion and from finally entering senescence. The fade of telomere shortening under pre‐ to postmenopausal estrogen concentrations suggests, at least in part, a senescence‐dependent cause for the onset of osteoarthritis in women after menopause. © 2011 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 29: 1563–1571, 2011     

自体骨髓间充质干细胞复合壳聚糖/羟基磷灰石支架修复兔膝骨软骨缺损

目的 探讨骨髓间充质干细胞(bone mesenehymal stem cells,BMSCs)复合壳聚糖(chitosan,CS)/羟基磷灰石(hydmxyapatite,HA)支架修复兔膝关节局部骨软骨缺损.方法 选健康日本大耳白兔36只,2～3月龄,体重1.7～2.0 kg,每只抽取自体骨髓4～6ml,体外分离培养BMSCs后以2×107/ml密度植于CS/HA支架上体外培养10 h,制成BMSCs-CS/HA支架复合物.将36只实验动物手术制成右膝股骨外侧髁负重区骨缺损模型后,随机分成A、B、C 3组,每组12只.A组植入BMSCs-CS/HA复合物,B组植入单纯CS/HA支架;C组不作任何植入,为空白对照组.分别于术后6周、12周各处死6只动物,取材后进行大体、组织学观察6根据改良Wakitani评分标准进行评分,评估软骨组织的修复情况,并行成组设计方差分析.结果 A组术后6周即可重建关节软骨缺损;修复软骨在观察期内逐渐变厚,软骨下骨有少量骨修复;术后12周透明软骨样修复,表面光整,与周围软骨色泽相近,软骨下骨有部分修复.而B组和C组12周时缺损区仍为纤维软骨样纤维组织修复,色泽浅黄.术后6、12周各组组织学半定量评分显示:股骨髁负重区修复A组评分明显优于B、C组(F=27.26,P＜0.05).结论 自体BMSCs复合CS/HA支架在体内环境下可形成透明软骨修复兔膝关节负重区骨软骨缺损.