Arthroscopic Shaver-based Harvest of Minced Cartilage Results in Reduced Chondrocyte Viability and Reduced Quality of Cartilaginous Repair Tissue Compared With Open Harvest and Conventional Fragmentation.

PURPOSE: To characterize and compare the quality of regenerative cartilage tissue (ReCT) after conventional minced cartilage (CMC) and arthroscopic minced cartilage (AMC), in terms of cell viability, gene expression, and matrix synthesis and to investigate the influence of different shaver types. METHODS: Chondral tissue was harvested from the knees of 8 porcine donors. Porcine specimens were euthanized one day before harvest. AMC was created with 2 shaver blades in 2 operating modes (oscillating vs forward) and compared with a scalpel-fragmented CMC control. Before histologic analysis, 50% of the tissue was digested to prevent dedifferentiation of chondrocytes to fibroblasts. Cells were cultured and analyzed for cell viability, gene expression of cartilage-specific markers (aggrecan [ACAN], collagen type II, alpha1 [COL2A1], collagen type I, alpha1 [COL1A1], fibronectin-1 [FN1]), and matrix synthesis (Alcian-blue). RESULTS: AMC tissue contained fewer viable chondrocytes (41%-54% vs 91%; P = .001-.048) compared with CMC. After culture, CMC showed greater expressions of ACAN (27 virtual copy numbers [VCN]/housekeeping gene) and COL2A1 (30 VCN) compared with AMC (ACAN 2-9 VCN, COL2A1 2-7 VCN, P = .001-.039). AMC presented greater expressions of COL1A1 (9-21 VCN) and FN1 (12-17 VCN) than CMC (1 and 6 VCN, P = .001-.050). The signal intensity of the cartilage matrix formed by CMC (86/mm2) was greater than by AMC (7-10 mm2, P = .001-.032). CONCLUSIONS: CMC contained high numbers of viable chondrocytes, resulting in high-quality, hyaline-like ReCT. In contrast, AMC showed impaired chondrocyte quantity and viability, showing greater expressions of fibroblast markers and a decreased formation of mature cartilage matrix in porcine samples. The high chondrogenic potential of CMC to form hyaline-like ReCT was not confirmed for AMC. CLINICAL RELEVANCE: On the basis of our findings, arthroscopic harvest of minced cartilage leads to reduced chondrocyte viability and ReCT quality. Accordingly, CMC and AMC cannot be regarded as synonymous techniques, as arthroscopic techniques seem to be less efficacious.

ACL reconstruction demonstrates better stability compared to ACL repair for patients with Schenck III and IV knee dislocations.

PURPOSE: This study compared mid-term outcomes of two anterior cruciate ligament (ACL) restoration techniques within an early total surgical care of acute knee dislocation: repair with additional internal bracing (ACLIB) and reconstruction with autograft (ACLR). Initial results at 12 months demonstrated that ACLR offered superior stability compared to ACLIB. METHODS: Retrospective clinical study of patients with acute type III or IV KD. ACLIB or ACLR procedures were performed accompanied by simultaneous suture and internal bracing of the posterior cruciate ligament (PCL) and repair with lateral augmentation of the medial and lateral complex injuries utilizing Arciero's reconstruction technique. Patient-reported outcome measurements (PROMs), instrumental stability assessment via the Rolimeter-Test, and stress radiographs were analyzed. RESULTS: The study involved 20 patients (5 IIIM, 5 IIIL, and 10 IV injuries) with an average follow-up of 35.2 ± 7.4 months. Notable differences in anterior tibial translation on stress radiography favouring ACLR persisted at 24-month follow-up in side-to-side difference (SSD) (ACLIB 2.8 ± 2.5 mm vs. ACLR 0.3 ± 2.6 mm; p = 0.0487), but Rolimeter test variance diminished (SSD ACLIB 2.5 ± 0.9 mm vs. ACLR 1.8 ± 1.7 mm). Both groups showed excellent PROMs (Lysholm Score: ACLIB 84.4 ± 15.8 vs. ACLR 89.9 ± 9.0; IKDC Score: ACLIB 77.1 ± 16.2 vs. ACLR 77.7 ± 8.6). CONCLUSION: Our results indicate improved anterior stability at 12 months, which persisted at 24 months after ACL reconstruction compared with ACL repair by stress radiography. Both groups showed favourable patient-reported outcomes throughout the follow-up period. Notable rates of postoperative knee stiffness were observed in both groups. These were successfully managed with early, one-time arthroscopic arthrolysis within the first seven months of treatment, resulting in no major range of motion limitations at the 24-month follow-up. LEVEL OF EVIDENCE: Retrospective cohort study, III.

Analysis of postoperative complications 5 years after osteosynthesis of patella fractures-a retrospective, multicenter cohort study.

PURPOSE: The study aims to investigate the influence of patient- and fracture-specific factors on the occurrence of complications after osteosynthesis of patella fractures and to compare knee joint function, activity, and subjective pain levels after a regular postoperative course and after complications in the medium term. METHODS: This retrospective, multicenter cohort study examined patients who received surgery for patella fracture at level 1 trauma centers between 2013 and 2018. Patient demographics and fracture-specific variables were evaluated. Final follow-up assessments included patient-reported pain scores (NRS), subjective activity and knee function scores (Tegner Activity Scale, Lysholm score, IKDC score), complications, and revisions. RESULTS: A total of 243 patients with a mean follow-up of 63.4 ± 21.3 months were included. Among them, 66.9% of patients underwent tension band wiring (TBW), 19.0% received locking plate osteosynthesis (LPO), and 14.1% underwent screw osteosynthesis (SO). A total of 38 patients (15.6%) experienced complications (TBW: 16.7%; LPO: 15.2%; SO: 11.8%). Implant-related complications of atraumatic fragment dislocation and material insufficiency/dislocation, accounted for 50% of all complications, were significantly more common after TBW than LPO (p = 0.015). No patient-specific factor was identified as a general cause for increased complications. Overall, particularly following complications such as limited range of motion or traumatic refracture, functional knee scores were significantly lower and pain levels were significantly higher at the final follow-up when a complication occurred. Implant-related complications, however, achieved functional scores comparable to a regular postoperative course without complications after revision surgery. CONCLUSION: The present study demonstrated that implant-related complications occurred significantly more often after TBW compared to LPO. The complication rates were similar in all groups.

Influence of different CCD angles on osseointegration and radiological changes after total hip arthroplasty of a triple wedge shape cementless femoral stem: a prospective cohort study.

PURPOSE: The purpose of this study was to evaluate the osseointegration and radiological outcomes in patients after total hip arthroplasty, hypothesizing different load patterns with one cementless stem design and different CCD angles (CLS Spotorno femoral stem 125° vs 135°). METHODS: All cases of degenerative hip osteoarthritis fulfilling strict inclusion criteria were treated with cementless hip arthroplasty between 2008 and 2017. Ninety-two out of one hundred six cases were clinically and radiologically examined three and 12 months after implantation. Two groups with each 46 patients were rendered prospectively and compared in clinical (Harris Hip Score) and radiological outcome. RESULTS: At final follow-up, no significant difference regarding Harris Hip Score was detected between the two groups (mean 99.2 ± 3.7 vs. 99.3 ± 2.5; p = 0.73). Cortical hypertrophy was found in none of the patients. Stress shielding was seen in a total of 52 hips (n = 27 vs. n = 25; 57% of the 92 hips). No significant difference regarding stress shielding was detected when comparing both groups (p = 0.67). Significant bone density loss was detected in Gruen zone one and two in the 125° group. The 135° group showed significant radiolucency in Gruen zone seven. No overall radiological loosening or subsidence of the femoral component was observed. CONCLUSION: According to our results, the use of a femoral component with a 125° CCD angle versus a 135° CCD did not result in a different osseointegration and load transfer with a clinically relevant significance.

Anterolateral versus modified posterolateral approach for tibial plateau fractures with involvement of the posterior column: a cadaveric study.

INTRODUCTION: The aim of this study was to compare the reduction quality of the anterolateral (AL) and modified posterolateral approach (PL) in lateral tibial plateau fractures involving the posterior column and central segments. METHODS: Matched pairs of pre-fractured cadaveric tibial plateau fractures were treated by either AL approach (supine position) or PL approach (prone position). Reduction was controlled by fluoroscopy and evaluated as satisfying or unacceptable. Afterwards, the reduction was examined by 3D scan. RESULTS: 10 specimens (3 pairs 41B3.1, 2 pairs 41C3.3) were evaluated. PL approach achieved significantly (p 0.00472) better fracture reduction results (0.4 ± 0.7 mm) of the posterior column compared to the AL group (2.1 ± 1.4 mm). Fracture steps involving the central area of the lateral plateau were insufficiently reduced after fluoroscopy using both approaches. CONCLUSION: Optimal reduction of displaced tibial plateau fractures involving the posterolateral column necessitates a posterior approach, which can be conducted in prone or lateral positioning. The anterolateral approach is indicated in fractures with minor displacement of the posterolateral rim but fracture extension in the latero-central segments. In these cases, an additional video-assisted reduction or extended approaches are helpful.

[Change in the treatment of patellar fractures]. / Wandel in der Behandlung der Patellafrakturen.

BACKGROUND: Fractures of the patella can lead to substantial functional impairments due to its crucial function in the extensor mechanism of the knee joint. Patellar fractures are associated with a comparatively high complication rate depending on the osteosynthesis procedure used. Despite established diagnostics and various osteosynthesis procedures they are still a therapeutic challenge. OBJECTIVE: This article gives an overview of the change in the treatment of patellar fractures and highlights the importance of comprehensive diagnostics and fracture-adapted treatment, especially for the increasing fractures in old age. MATERIAL AND METHODS: Evaluation of the current literature, discussion of biomechanical and clinical studies and expert recommendations (guidelines) RESULTS: Tension band wiring is still the most commonly used procedure; however, it is associated with a high rate of implant-related complications and failure rates. In particular, multifragmented comminuted fractures can often not be adequately treated by tension band wiring. Anterior locking plates seem to be biomechanically and clinically superior. CONCLUSION: For a good functional result after a patellar fracture, the fracture morphology must be completely understood and the optimal choice of the osteosynthesis method is crucial. Distal (or proximal) pole fractures and the increasing number of osteoporotic fractures of old patients due to the demographic change, remain a therapeutic challenge and require special consideration in the selection of the osteosynthesis procedure.

[Current treatment standard for patella fractures in Germany]. / Aktueller Versorgungsstandard von Patellafrakturen in Deutschland.

BACKGROUND: The treatment of patella fractures is technically demanding. Although the radiological results are mostly satisfactory, this often does not correspond to the subjective assessment of the patients. The classical treatment with tension band wiring with K­wires has several complications. Fixed-angle plate osteosynthesis seems to be biomechanically advantageous. OBJECTIVE: Who is treating patella fractures in Germany? What is the current standard of treatment? Have modern forms of osteosynthesis become established? What are the most important complications? MATERIAL AND METHODS: The members of the German Society for Orthopedics and Trauma Surgery and the German Knee Society were asked to participate in an online survey. RESULTS: A total of 511 completed questionnaires were evaluated. Most of the respondents are specialized in trauma surgery (51.5%), have many years of professional experience and work in trauma centers. Of the surgeons 50% treat ≤5 patella fractures annually. In almost 40% of the cases preoperative imaging is supplemented by computed tomography. The classical tension band wiring with K­wires is still the preferred form of osteosynthesis for all types of fractures (transverse fractures 52%, comminuted fractures 40%). In the case of comminuted fractures 30% of the surgeons choose fixed-angle plate osteosynthesis. If the inferior pole is involved a McLaughlin cerclage is used for additional protection in 60% of the cases. DISCUSSION: The standard of care for patella fractures in Germany largely corresponds to the updated S2e guidelines. Tension band wiring is still the treatment of choice. Further (long-term) clinical studies are needed to verify the advantages of fixed-angle plates.

Bifunctional Labeling of Rabbit Mesenchymal Stem Cells for MR Imaging and Fluorescence Microscopy.

PURPOSE: Longitudinal imaging studies are important in the translational process of stem cell-based therapies. Small animal imaging models are widely available and practical but insufficiently depict important morphologic detail. In contrary, large animal models are logistically challenging and costly but offer greater imaging quality. In order to combine the advantages of both, we developed an intermediate-sized rabbit animal model for cartilage imaging studies. PROCEDURES: Rabbit mesenchymal stem cells (rMSC) were isolated as primary cultures from the bone marrow of New Zealand white rabbits. rMSC were subsequentially transduced lentivirally with eGFP and magnetically labeled with the iron oxide ferucarbotran. eGFP expression was evaluated by flow cytometry and iron uptake was analyzed by isotope dilution mass spectrometry and Prussian blue staining. Fluorescence microscopy of eGFP-transduced rMSC was performed. Viability and induction of apoptosis were assessed by XTT and caspase-3/-7 measurements. The chondrogenic potential of labeled cells was quantified by glycosaminoglycan contents in TGF-ß3 induced pellet cultures. Labeled and unlabeled cells underwent magnetic resonance imaging (MRI) at 1.5 T before and after differentiation using T1-, T2-, and T2*-weighted pulse sequences. Relaxation rates were calculated. rMSCs were implanted in fibrin clots in osteochondral defects of cadaveric rabbit knees and imaged by 7 T MRI. T2* maps were calculated. Statistical analyses were performed using multiple regression models. RESULTS: Efficiency of lentiviral transduction was greater than 90 %. Fluorescence signal was dose dependent. Cellular iron uptake was significant for all concentrations (p < 0.05) and dose dependent (3.3-56.5 pg Fe/cell). Labeled rMSC showed a strong, dose-dependent contrast on all MR pulse sequences and a significant decrease in T2 and T2* relaxation rates. Compared with non-transduced or unlabeled controls, there were no adverse effects on cell viability, rate of apoptosis, or chondrogenic differentiation. MRI of labeled rMSCs in osteochondral defects showed a significant signal of the transplant with additional high-resolution anatomical information. CONCLUSIONS: This intermediate-sized rabbit model and its bifunctional labeling technique allow for improved depiction of anatomic detail for noninvasive in vivo rMSC tracking with MRI and for immunohistological correlation by fluorescence microscopy.

Chondrocyte Culture Parameters for Matrix-Assisted Autologous Chondrocyte Implantation Affect Catabolism and Inflammation in a Rabbit Model.

Cartilage defects represent an increasing pathology among active individuals that affects the ability to contribute to sports and daily life. Cell therapy, such as autologous chondrocyte implantation (ACI), is a widespread option to treat larger cartilage defects still lacking standardization of in vitro cell culture parameters. We hypothesize that mRNA expression of cytokines and proteases before and after ACI is influenced by in vitro parameters: cell-passage, cell-density and membrane-holding time. Knee joint articular chondrocytes, harvested from rabbits (n = 60), were cultured/processed under varying conditions: after three different cell-passages (P1, P3, and P5), cells were seeded on 3D collagen matrices (approximately 25 mm³) at three different densities (2 × 105/matrix, 1 × 106/matrix, and 3 × 106/matrix) combined with two different membrane-holding times (5 h and two weeks) prior autologous transplantation. Those combinations resulted in 18 different in vivo experimental groups. Two defects/knee/animal were created in the trochlear groove (defect dimension: ∅ 4 mm × 2 mm). Four identical cell-seeded matrices (CSM) were assembled and grouped in two pairs: One pair giving pre-operative in vitro data (CSM-i), the other pair was implanted in vivo and harvested 12 weeks post-implantation (CSM-e). CSMs were analyzed for TNF-α, IL-1ß, MMP-1, and MMP-3 via qPCR. CSM-i showed higher expression of IL-1ß, MMP-1, and MMP-3 compared to CSM-e. TNF-α expression was higher in CSM-e. Linearity between CSM-i and CSM-e values was found, except for TNF-α. IL-1ß expression was higher in CSM-i at higher passage and longer membrane-holding time. IL-1ß expression decreased with prolonged membrane-holding time in CSM-e. For TNF-α, the reverse was true. Lower cell-passages and lower membrane-holding time resulted in stronger TNF-α expression. Prolonged membrane-holding time resulted in increased MMP levels among CSM-i and CSM-e. Cellular density was of no significant effect. We demonstrated cytokine and MMP expression levels to be directly influenced by in vitro culture settings in ACI. Linearity of expression-patterns between CSM-i and CSM-e may predict ACI regeneration outcome in vivo. Cytokine/protease interaction within the regenerate tissue could be guided via adjusting in vitro culture parameters, of which membrane-holding time resulted the most relevant one.

Primary Total Hip Arthroplasty in Severe Dysplastic Hip Osteoarthritis With a Far Proximal Cup Position.

BACKGROUND: Developmental hip dysplasia is the most common cause of secondary hip osteoarthritis. Due to severe acetabular bone deficiency, cup positioning in total hip arthroplasty (THA) of dysplastic hips remains a surgical challenge. The aim was to analyze the functional outcome of far proximal cup positions in primary THA. METHODS: Fifty patients (61 hips) with THA for severe dysplastic osteoarthritis and a far proximal cup position were included. Patients were divided according to the heights of the implanted cups with increasing vertical distance from the interteardrop line (group A: 55-65 mm, group B: 65-75 mm, group C: >75 mm). Functional outcome was assessed at latest follow-up (38 ± 16 months) by Lower Extremity Functional Score, Tegner Activity Score, and Harris Hip Score (HHS). Patients answered a Patient Satisfaction Questionnaire. Leg length discrepancy was estimated radiographically. RESULTS: The Lower Extremity Functional Score significantly decreased in C (45.3 ± 25) compared to A (66.7 ± 15.3) and B (67.9 ± 9.9). The Tegner Activity Score significantly increased in all subgroups from preoperative to postoperative (2.2 ± 1.3 to 4.1 ± 1.4; P < .05). The mean overall HHS was 89.3 ± 14.7 (A: 89.5 ± 14.3, B: 94.3 ± 6.5, C: 78.3 ± 22.1). The HHS domains of activity of daily life and gait were significantly reduced in C (P < .05). Patients described a high satisfaction level with the surgery. No significant differences were found with regard to preoperative and postoperative leg lengthening (P = .881). Neither dislocations, impingement problems nor neurologic complications were observed. CONCLUSION: Primary THA without any concomitant surgical interventions with a far proximal cup position offers a safe and effective treatment option in severe dysplastic hip osteoarthritis.

Detection of intramyocardially injected DiR-labeled mesenchymal stem cells by optical and optoacoustic tomography.

The distribution of intramyocardially injected rabbit MSCs, labeled with the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbo-cyanine-iodide (DiR) using hybrid Fluorescence Molecular Tomography-X-ray Computed Tomography (FMT-XCT) and Multispectral Optoacoustic Tomography (MSOT) imaging technologies, was investigated. Viability and induction of apoptosis of DiR labeled MSCs were assessed by XTT- and Caspase-3/-7-testing in vitro. 2 × 106, 2 × 105 and 2 × 104 MSCs labeled with 5 and 10 µg DiR/ml were injected into fresh frozen rabbit hearts. FMT-XCT, MSOT and fluorescence cryosection imaging were performed. Concentrations up to 10 µg DiR/ml did not cause apoptosis in vitro (p > 0.05). FMT and MSOT imaging of labeled MSCs led to a strong signal. The imaging modalities highlighted a difference in cell distribution and concentration correlated to the number of injected cells. Ex-vivo cryosectioning confirmed the molecular fluorescence signal. FMT and MSOT are sensitive imaging techniques offering high-anatomic resolution in terms of detection and distribution of intramyocardially injected stem cells in a rabbit model.

Fluorescence molecular tomography of DiR-labeled mesenchymal stem cell implants for osteochondral defect repair in rabbit knees.

OBJECTIVES: To assess labelling efficiency of rabbit mesenchymal stem cells (MSCs) using the near-infrared dye 1,1'-dioctadecyl-3,3,3',3'-tetramethylindotricarbocyanine iodide (DiR) and detection of labelled MSCs for osteochondral defect repair in a rabbit model using fluorescence molecular tomography-X-ray computed tomography (FMT-XCT). METHODS: MSCs were isolated from New Zealand White rabbits and labelled with DiR (1.25-20 µg/mL). Viability and induction of apoptosis were assessed by XTT- and Caspase-3/-7-testing. Chondrogenic potential was evaluated by measurement of glycosaminoglycans. Labelled cells and unlabeled controls (n = 3) underwent FMT-XCT imaging before and after chondrogenic differentiation. Osteochondral defects were created surgically in rabbit knees (n = 6). Unlabeled and labelled MSCs were implanted in fibrin-clots and imaged by FMT-XCT. Statistical analyses were performed using multiple regression models. RESULTS: DiR-labelling of MSCs resulted in a dose-dependent fluorescence signal on planar images in trans-illumination mode. No significant reduction in viability or induction of apoptosis was detected at concentrations below 10 µg DiR/mL (p > .05); the chondrogenic potential of MSCs was not affected (p > .05). FMT-XCT of labelled MSCs in osteochondral defects showed a significant signal of the transplant (p < .05) with additional high-resolution anatomical information about its osteochondral integration. CONCLUSIONS: FMT-XCT allows for detection of stem cell implantation within osteochondral regeneration processes. KEY POINTS: • DiR-labelling of MSCs shows no toxic side effects or impairment of chondrogenesis. • Fluorescence molecular tomography allows for detection of MSCs for osteochondral defect repair. • FMT-XCT helps to improve evaluation of cell implantation and osteochondral regeneration processes.

Matrix-assisted autologous chondrocyte transplantation for remodeling and repair of chondral defects in a rabbit model.

Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration (1). Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery (2-4). Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies. The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) (5). Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation (6). In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular (7-9). MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration (10). The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics (11). The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit's knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.

Treatment of osteochondral defects in the rabbit's knee joint by implantation of allogeneic mesenchymal stem cells in fibrin clots.

The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface (1). Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential (2). In the last decades, several surgical treatment options have emerged and have already been clinically established (3-6). Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface (3,7,8). Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects. New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation (9,10). However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone (11). The sandwich-technique combines bone grafting with current approaches in Tissue Engineering (5,6). This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing (12). Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity (11). Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential (13,14). The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect. In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results (1,15-18). Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies (19-21) and even first human trials (22). The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit's bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit's knee joint will be described.

The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration.

Matrix-assisted chondrocyte transplantation (m-ACI) still lacks any standardization in its execution in terms of cell passage (P), cell yield (C) and in vitro membrane-holding time (T). It was the goal of this study to analyze the effect of shifting cell culture parameters (P, C, T) on the in vitro as well as in vivo effort of a regulated animal m-ACI. Autologous rabbit knee articular chondrocytes were seeded within bilayer collagen I/III 3-D matrices in variation of P, C and T. Each time, 2 PCT-identical by 2 PCT-identical cell-matrix-constructs (CMC)/animal were created. Simultaneously 2 (PCT-distinct) were re-implanted (CMC-e) autologous into artificial trochlear pristine chondral defects in vivo to remain for 12 weeks while the remaining 2 were harvested (CMC-i) for immediate in vitro analysis at the time of transplantation of their identical twins. mRNA of both, CMC-e regenerates and CMC-i membranes, was analyzed for Collagen-1,-2,-10, COMP, Aggrecan, Sox9 expression by use of a mixed linear model, multiple regression analysis. Generally, CMC-i values were higher than CMC-e values for differentiation targets; the opposite was true for dedifferentiation targets. Regarding individual gene expression, in vivo regenerate cell-matrix properties were significantly dependent on initial cell-matrix in vitro values as a sign of linearity. The parameter membrane-holding time (T) had strongest effects on the resulting mRNA expression with slightly less impact of the parameter passage (P), whereas cell yield (C) had clearly less effects. Noting differences between in vitro and in vivo data, in general, optimal expression patterns concerning chondrogenic differentiation were achieved by few passages, medium cellular yield, short membrane-holding time. Clinical m-ACI may benefit from optimal orchestration of the cell culture parameters passage, yield and time.