Repair of human articular cartilage after implantation of autologous chondrocytes

Tissue engineering is an increasingly popular method of addressing pathological disorders of cartilage. Recent studies have demonstrated its clinical efficacy, but there is little information on the structural organisation and biochemical composition of the repair tissue and its relation to the adjacent normal tissue. We therefore analysed by polarised light microscopy and immunohistochemistry biopsies of repair tissue which had been taken 12 months after implantation of autologous chondrocytes in two patients with defects of articular cartilage. Our findings showed zonal heterogeneity throughout the repair tissue. The deeper zone resembled hyaline-like articular cartilage whereas the upper zone was more fibrocartilaginous. The results indicate that within 12 months autologous chondrocyte implantation successfully produces replacement cartilage tissue, a major part of which resembles normal hyaline cartilage.     

Quantitative analysis of gene expression in human articular chondrocytes assigned for autologous implantation

Autologous chondrocyte implantation (ACI) relies on the implantation of in vitro expanded cells. The aim was to study the dedifferentiation of human articular chondrocytes under different cultivating conditions [days 0–10 in the primary culture (P0); passages in a monolayer from P0 to P3; monolayer vs. alginate and monolayer vs. alginate/agarose hydrogels] using real‐time PCR analysis. The relative gene expressions for collagen type I and II, aggrecan and versican were quantified and the corresponding differentiation indexes (Col2/Col1, Agr/Ver) were calculated. The values of both differentiation indexes decreased exponentially with time in the P0 monolayer culture, and continued with a significant decrease over the subsequent monolayer passages. On the contrary, the chondrocytes seeded in either of the hydrogels significantly increased the indexes compared to their parallel monolayer cultures. These results indicate that alginate and alginate/agarose hydrogels offer an appropriate environment for human articular chondrocytes to redifferentiate after being expanded in vitro. Therefore the three‐dimensional (3D) hydrogel chondrocyte cultures present not only surgical, but also biological advantage over the classic suspension–periosteum chondrocyte implantation. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:847–853, 2008     

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。     

生长因子对成人关节软骨细胞的促增殖作用

目的观察不同生长因子对成人关节软骨细胞（adult human articular chondrocytes，AHAC）增殖的影响，探索AHAC体外大量扩增的方法。方法以酶消化法从成人关节软骨分离细胞，条件培养基培养；传2代细胞分别用不同浓度成纤维细胞生长因子2（fibroblast growth factor-2，FGF-2）、转化生长因子β1（transforming growth factor β1，TGF—β1）、血小板衍生因子bb（platelet derived growth factor-bb，PDGF-hb）、肝细胞生长因子（hepatocyte growth factor，HGF）或其不同组合作用。用MTT法比较细胞增殖情况，用组化和免疫组化检测观察细胞表型变化。结果FGF-2、TGF—β1、PDGF—bb、HGF均有促AHAC增殖的作用，其最大效应剂量分别是50ng/ml、1ng／ml、1ng／ml、20ng／ml。5ng／ml FGF-2＋1ng/ml TGF-β1有最强的促增殖作用，继续加用PDGF—bb和（或）HGF无进一步促进作用；用这一因子组合培养AHAC，可以传10代以上，细胞扩增2000倍以上，且传9代细胞仍弱表达Ⅱ型胶原和aggrecan。结论FGF-2、TGF-β1、PDGF—bb、HGF均对AHAC有一定的促增殖作用；5ng／ml FGF-2＋1ng/ml TGF-β1有最大的促增殖效应，细胞短期内大量扩增，且在大量扩增的同时维持了一定的软骨细胞表型，因此是合适的AHAC体外大量扩增促进剂。     

基质诱导的自体软骨细胞移植术修复膝关节软骨缺损1O例术后2年的随访

目的应用基质诱导的自体软骨细胞移植技术（MACI,Genzyme,America）对膝关节软骨损伤的患者进行治疗,通过对患者进行术后2年的随访分析,评价MACI治疗的安全性和有效性。方法从2004年至2008年11月,对10例患者实施MACI手术。患者平均年龄34.9岁（14～57岁）,缺损的平均面积（3.69±2.62）cm2/处（0.4～8cm2,n=20）。MACI技术是从患者非负重区取自体关节软骨,进行体外消化并分离出软骨细胞,培养扩增后接种在Ⅰ/Ⅲ型双层胶原膜上。移植前按缺损的部位修剪成相应形状,用生物蛋白胶将胶原膜粘贴在关节软骨缺损处。分别于术前1周、术后3个月、6个月、1年和2年使用KOOS评分问卷进行临床康复效果的评估并进行术后核磁共振的检测（MRI）。此外,术后对2例患者进行了3次关节镜检查和2次组织学检测。结果 10例患者术后均未发生并发症及与手术相关的不良事件。患者术后3个月KOOS评分显示疼痛明显改善（P〈0.05）;术后6个月KOOS评分显示：疼痛、症状、日常生活、运动及娱乐和生活质量5项均明显升高,5项评分间有统计学差异（P〈0.05）。术后1年和2年患者的情况得到了持续的改善（P〈0.05）。患者术后3个月的MRI显示软骨缺损部位得到大部分填充和修复;6个月移植软骨基本与周围软骨完全整合;1年后修复组织呈等信号,软骨下骨髓水肿消失;2年后大部分患者软骨修复组织信号与周围组织信号强度一致,软骨下骨无骨髓水肿。术后15个月和2年的组织学检查显示新生的软骨组织以透明软骨为主。MACI手术一般都能在2h内完成,术中出血量小于100ml。结论 MACI技术是修复关节软骨缺损安全、可靠和有效的治疗措施,具有操作简单、手术时间短和术中出血量少等特点。     

In vitro response of human chondrocytes to a combination of growth factors and a proteinase inhibitor

Degenerative disk disease is an accelerating cascade of tissue degeneration in the intervertebral disk. A harsh catabolic environment perpetuates the degeneration of the intervertebral disk. Tissue engineering-based techniques offer effective treatment to slow the progression of degenerative disk disease and regenerate intervertebral disk tissue. The purpose of this study was to assess the efficacy of a regenerative therapy for degenerative disk disease by treating human chondrocytes with anabolic growth factors and a proteinase inhibitor. The use of both proved effective in upregulating important extracellular matrix markers of human chondrocytes. These successful in vitro results have implications for the regeneration of the intervertebral disk.     

The arthroscopic implantation of autologous chondrocytes for the treatment of full-thickness cartilage defects of the knee joint

Autologous chondrocyte implantation is an established option for the treatment of full-thickness cartilage defects of the knee. Open implantation has a high morbidity. On a resorbable polymer fleece, autologous chondrocytes can be implanted arthroscopically. Transosseous anchoring assures high initial stability of the implant. Tibial defects can be addressed. The arthroscopic technique for the implantation of autologous chondrocytes eliminates a substantial amount of the side effects known to occur after open autologous chondrocyte implantation procedures.     

In vitro cell quality of articular chondrocytes assigned for autologous implantation in dependence of specific patient characteristics

Objective  

Autologous chondrocyte implantation (ACI) is a well-established therapeutic option for the treatment of cartilage defects of the knee joint. Since information concerning the cellular aspects of ACI is still limited, the aim of the present study was to investigate relevant differences between chondrocyte quality after in vitro cultivation and possible correlations with patient-specific factors.     

Viability of chondrocytes seeded onto a collagen I/III membrane for matrix‐induced autologous chondrocyte implantation

Cell viability is crucial for effective cell‐based cartilage repair. The aim of this study was to determine the effect of handling the membrane during matrix‐induced autologous chondrocyte implantation surgery on the viability of implanted chondrocytes. Images were acquired under five conditions: (i) Pre‐operative; (ii) Handled during surgery; (iii) Cut edge; (iv) Thumb pressure applied; (v) Heavily grasped with forceps. Live and dead cell stains were used. Images were obtained for cell counting and morphology. Mean cell density was 6.60 × 10⁵ cells/cm² (5.74–7.11 × 10⁵) in specimens that did not have significant trauma decreasing significantly in specimens that had been grasped with forceps (p < 0.001) or cut (p = 0.004). Cell viability on delivery grade membrane was 75.1%(72.4–77.8%). This dropped to 67.4%(64.1–69.7%) after handling (p = 0.002), 56.3%(51.5–61.6%) after being thumbed (p < 0.001) and 28.8%(24.7–31.2%) after crushing with forceps (p < 0.001). When cut with scissors there was a band of cell death approximately 275 µm in width where cell viability decreased to 13.7%(10.2–18.2%, p < 0.001). Higher magnification revealed cells without the typical rounded appearance of chondrocytes. We found that confocal laser‐scanning microscope (CLSM) can be used to quantify and image the fine morphology of cells on a matrix‐induced autologous chondrocyte implantation (MACI) membrane. Careful handling of the membrane is essential to minimise chondrocyte death during surgery. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:1495–1502, 2014.     

Characteristics of cartilage biopsies used for autologous chondrocytes transplantation

Biopsies removed from 57 patients considered for cartilage transplantation were grown at CTI Ltd. (47 biopsies) and at Tel Aviv University (10 biopsies). Tissue processing took place in dedicated laboratories. Explant cultures allowed cell number expansion. Fifty-four out of 57 biopsies grew cells. Fanning out of the cells began after 5-15 days in culture. Two passages later, cell numbers in the 10(7) range were achieved. Cells from all cultures expressed mRNA of aggrecan and link protein but not of alkaline phosphatase. Histochemical stains such as alcian blue pH 1 were negative in sparse monolayer cultures, but positive in pellet cultures. Immunohistochemistry demonstrated expression of collagen type I in monolayer cultures, switching to collagen type II in micromass cultures. Fibroblast growth factor receptor 3, a recently described characteristic receptor of precartilaginous cells, was expressed in monolayers and disappeared in micromass cultures. In conclusion, explants of articular chondrocytes cultured in vitro consistently yield monolayer cultures. The cells appear to revert to dedifferentiated chondrocytes, expressing a mesenchymal stem cell protein profile. Simultaneously, these cells regained their capacity to proliferate. Cultures held as micromass allowed reexpression of the differentiated phenotype traits.     

Responsiveness of bovine chondrocytes to growth factors in medium with different serum concentrations.

Autologous transplantation of chondrocytes is currently under investigation as a potential therapy to stimulate intrinsic repair in articular cartilage defects. The quality of the repair tissue may benefit from the preservation of the characteristic chondrocytic phenotype of the transplanted cells together with the production of a new extracellular matrix composed of collagen type II and larger proteoglycans. A number of growth factors are believed to play an important role in the process of generating new cartilage repair tissue. In this study, the dose-dependent response of bovine chondrocytes to recombinant human insulin-like growth factor-1, recombinant human transforming growth factor-beta2, and recombinant human bone morphogenetic protein-2 was studied in an alginate culture system under different culture conditions. The chondrocytes were cultured in medium with increasing concentrations of fetal calf serum. The cultures were assessed by the total amount of DNA, quantitative and qualitative synthesis of proteoglycan, production of nitric oxide, and histology. Cells cultured in the presence of each growth factor had an equal, nonsignificant stimulation of DNA synthesis compared with those cultured in basal medium alone. Recombinant human insulin-like growth factor-1 and recombinant human transforming growth factor-beta2 stimulated proteoglycan synthesis in a dose-dependent and reversed dose-dependent fashion, respectively. Recombinant human bone morphogenetic protein-2 stimulated proteoglycan synthesis significantly only in the absence of fetal calf serum or in the presence of small amounts of the serum. Overall, proteoglycan synthesis dramatically decreased with the addition of each growth factor as the concentration of fetal calf serum in the medium decreased, and the dose-dependent stimulation pattern, as observed for recombinant human insulin-like growth factor-1 and recombinant human transforming growth factor-beta2, disappeared. Apart from a moderate increase in mRNA for aggrecan and decorin, the growth factors did not greatly affect the type of proteoglycans synthesized. Histological examination confirmed the presence of a dense pericellular matrix deposition, especially when the chondrocytes were cultured in the presence of recombinant human insulin-like growth factor-1 or recombinant human transforming growth factor-beta2. The results indicate that these growth factors can stimulate qualitatively superior matrix production and that the responsiveness of the chondrocytes to the growth factors changes with the culture conditions. Further knowledge about the interaction between chondrocytes, growth factors, and the external environment is important to stimulate chondrocytes to produce adequate repair tissue in cartilage defects in vivo. Insulin-like growth factor-1 especially seems capable of stimulating, in the most consistent and predictable fashion, qualitatively superior proteoglycan synthesis by differentiated chondrocytes. Additional in vivo studies are needed to evaluate the potential of these growth factors as stimulators in cartilage repair.     

Cellular reactions to biodegradable magnesium alloys on human growth plate chondrocytes and osteoblasts

Purpose

In recent decades operative fracture treatment using elastic stable intramedullary nails (ESINs) has mainly taken precedence over conservative alternatives in children. The development of biodegradable materials that could be used for ESINs would be a further step towards treatment improvement. Due to its mechanical and elastic properties, magnesium seems to be an ideal material for biodegradable implant application. The aim of this study was therefore to investigate the cellular reaction to biodegradable magnesium implants in vitro.

Methods

Primary human growth plate chondrocytes and MG63 osteoblasts were used for this study. Viability and metabolic activity in response to the eluate of a rapidly and a slower degrading magnesium alloy were investigated. Furthermore, changes in gene expression were assessed and live cell imaging was performed.

Results

A superior performance of the slower degrading WZ21 alloy’s eluate was detected regarding cell viability and metabolic activity, cell proliferation and morphology. However, the ZX50 alloy’s eluate induced a favourable up-regulation of osteogenic markers in MG63 osteoblasts.

Conclusions

This study showed that magnesium alloys for use in biodegradable implant application are well tolerated in both osteoblasts and growth plate chondrocytes respectively.     

Basic fibroblast growth factor regulates expression of growth factors in rat epiphyseal chondrocytes.

Chondrocytes produce several local regulatory factors such as basic fibroblast growth factor (bFGF), transforming growth factor-beta (TGF-beta) and insulin-like growth factor-I (IGF-I). In this study, we examined the effect of bFGF on the expressions of both mRNA and protein of the growth factors synthesized by chondrocytes. Treatment of chondrocytes with bFGF (1-100 ng/ml) stimulated the mRNA expression of bFGF and TGF-beta up to 121-604% and 130-220% at 12 h compared with the controls, respectively. On the other hand, the treatment of chondrocytes with bFGF (1-100 ng/ml) suppressed IGF-I mRNA expression to 79-47% at 12 h compared with the controls. An enzyme-linked immunosorbent assay (ELISA) revealed that the treatment of chondrocytes with bFGF (1-100 ng/ml) also enhanced the production of TGF-beta proteins in the chondrocytes up to 299-508% at 24 h compared with controls. We conclude that bFGF influenced the local expression of growth factors by chondrocytes, suggesting autoregulation of growth factor expression during chondrogenesis.     

Clinical and cellbiological aspects of autologous chondrocytes transplantation

The treatment of deep cartilage defects is a challenge for every orthopaedic surgeon. The potential for regeneration of cartilage tissue is minimal and leads to mechanically inferior fibrous tissue. The established techniques induce the growth of fibrous tissue but fail to prevent arthrosis. Autologous chondrocyte transplantation seems to be the most promising therapy concept with clinical relevance to reserves a full thickness cartilage defect with hyaline-like cartilage. Outcome studies with a follow up from 2-10 years show in up to 90 % good and excellent results for defects on the femoral condyle and 70 % for the patella. Mechanical testing of the regenerated cartilage showed almost similar stiffness as nearly normal hyaline cartilage. The available data justify the acceptance of autologous chondrocyte transplantation as a standard procedure for limited indications and well-trained surgeons. Result of already inaugurated studies will show the potential of chondrocyte transplantation to prevent osteoarthritis.     

Cell-based therapy options for osteochondral defects. Autologous mesenchymal stem cells compared to autologous chondrocytes

Cartilage defects are multifactorial and site-specific and therefore need a clear analysis of the underlying pathology as well as an individualized therapy so that cartilage repair lacks a one-for-all therapy. The results of comparative clinical studies using cultured chondrocytes in autologous chondrocyte implantation (ACI) have shown some superiority over conventional microfracturing under defined conditions, especially for medium or large defects and in long-term durability. Adult mesenchymal stem cells can be isolated from bone marrow, have the potency to proliferate in culture and are capable of differentiating into the chondrogenic pathway. They represent a promising versatile cell source for cartilage repair but the ideal conditions for cultivation and application in cartilage repair are not yet known or have not yet been characterized. Adding a scaffold offers mechanical stability and advances chondrogenic differentiation for both possible cell sources.     

Response of human growth hormone to anesthesia induction with propofol

Although there are distinct differences in both pharmacokinetics and chemical structure, propofol has sedative effects similar to those of benzodiazepines. Both diazepam and thiopental, commonly used agents for inducing anaesthesia, show some typical neuroendocrine effects such as liberation of human growth hormone (hGH) in addition to their well-known influences on the cardiovascular system. It was our aim to examine the endocrine response of hGH after induction of anaesthesia with propofol and to compare any possible effects with those of diazepam and thiopental. The study was performed on 30 non-premedicated patients, who underwent plastic-surgery (mean age: x = 35 +/- 8.3 years, mean body weight: x = 68.9 +/- 23.2 kg). No signs of endocrine disturbances were found in the patients prior to the study. Patients were divided in a random fashion into 3 groups of 10 persons each: In group 1 propofol was given in a bolus injection (2 mg/kg) and then by infusion for a period of 10 min (0.2 mg/kg/min). In group 2 diazepam (0.3 mg/kg) and in group 3 thiopental (5 mg/kg) were administered as a bolus to induce anesthesia. All patients were intubated immediately after induction of anesthesia and then relaxed using vecuronium (1 mg/kg). Ventilation was performed mechanically during the entire operative period (N2O/O2 - FiO2:0.33, tidal volume 10 ml/kg, respiratory rate: 14/min). To maintain anesthesia halothane (0.5-1.0 vol.-%) and meperidine (0.75 mg/kg) were added 15 min after induction.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro analysis of differential expression of collagens, integrins, and growth factors in cultured human chondrocytes.

OBJECTIVES: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation and proliferation of the cells are critical factors that influence the generation of transplants. The aim of our study was to find and characterize markers for cell proliferation and dedifferentiation in cultured chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were isolated from septal cartilage and held in primary cell culture. Cells were harvested after 1, 6, and 21 days. The differentiation of the cells was investigated with bright-field microscopy, the expression patterns of various proteins using immunohistochemistry, and the expression of distinct genes with the microarray technique. RESULTS: The chondrocytes showed a strong proliferation. After 6 and 21 days, collagen 9 and 10 were downregulated; collagen 11 was activated. Collagen 1 and 2 were downregulated after 6 days but were reactivated after 21 days. Tumor growth factor beta (TGF-beta)1 was strongly expressed on days 1, 6, and 21, TGF-beta2 was never expressed, and TGF-beta3 and -beta4 were upregulated from day 1 to day 21. The TGF-beta receptor III was expressed on days 1, 6, and 21. Integrin beta1, beta5, and alpha5 were upregulated from day 1 to day 21; integrin beta3 was downregulated. CONCLUSION AND SIGNIFICANCE: Collagens 3, 4, 8, 9, and 11 might be new markers for the dedifferentiation of chondrocytes. Collagen 2 might be a marker for the synthetic activity of the cells rather than the dedifferentiation. TGF-beta3 and -beta4 might influence the dedifferentiation, which is fortified by the expression of TGF-beta receptor III. Integrin beta1, beta5, and alpha5 might be involved in signal transmission for the dedifferentiation.     

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.     

Do autologous growth factors enhance transforaminal lumbar interbody fusion?

Pseudarthrosis remains a significant problem in spinal fusion. The objective of our study was to investigate the effects of autologous growth factors (AGF) in instrumented transforaminal lumbar interbody spinal fusion (TLIF). A prospective review was carried out of 23 patients who underwent TLIF with application of AGF, with a minimum 2-year follow-up. Comparison with our historical cohort (without AGF application) was performed. Mean age at surgery was 44.3 years in the AGF treatment group. Twelve had a positive smoking history. Fourteen had undergone previous spinal surgeries. Thirteen received one-level fusions and ten received two-level fusions. The radiographic results showed a fusion rate of 100% in one-level fusions and 90% in two-level fusions. There was no significant difference in pseudarthrosis rates between the AGF treatment group and historical cohort. Excluding the cases with pseudarthrosis, there was faster bony healing in patients who had been treated with AGF application. This study indicates that although AGF may demonstrate faster fusions, it does not result in an overall increase in spinal fusion rates. Further studies are needed before AGF can routinely be used as an adjunct in spinal fusion.