Role of Matrix-Associated Autologous Chondrocyte Implantation with Spheroids in the Treatment of Large Chondral Defects in the Knee: A Systematic Review.

Autologous chondrocyte implantation (ACI) is a cell therapy for the treatment of focal cartilage defects. The ACI product that is currently approved for use in the European Union (EU) consists of spheroids of autologous matrix-associated chondrocytes. These spheroids are spherical aggregates of ex vivo expanded human autologous chondrocytes and their self-synthesized extracellular matrix. The aim is to provide an overview of the preclinical and nonclinical studies that have been performed to ensure reproducible quality, safety, and efficacy of the cell therapy, and to evaluate the clinical data on ACI with spheroids. A systematic review was performed to include all English publications on self-aggregated spheroids of chondrocytes cultured in autologous serum without other supplements. A total of 20 publications were included, 7 pre- and nonclinical and 13 clinical research publications. The pre- and nonclinical research publications describe the development from concept to in vivo efficacy and quality- and safety-related aspects such as biodistribution, tumorigenicity, genetic stability, and potency. The evaluation of clinical research shows short- to mid-term safety and efficacy for the ACI with spheroid-based treatment of cartilage defects in both randomized clinical trials with selected patients, as well as in routine treatment providing real-world data in more complex patients.

Effect of Human Serum and 2 Different Types of Platelet Concentrates on Human Meniscus Cell Migration, Proliferation, and Matrix Formation.

PURPOSE: To evaluate the effect of 10% human serum (HS), 5% platelet-rich plasma (PRP), and 5% autologous conditioned plasma (ACP) on migration, proliferation, and extracellular matrix (ECM) synthesis of human meniscus cells. METHODS: Cell migration and proliferation on stimulation with HS, PRP, and ACP were assessed by chemotaxis assays and measurement of genomic DNA content. Meniscus cells were cultivated in pellets stimulated with 10% HS, 5% PRP, or 5% ACP. Meniscal ECM formation was evaluated by histochemical staining of collagen type I, type II, and proteoglycans and by analysis of fibrochondrocyte marker gene expression. RESULTS: Human meniscus cells were significantly attracted by all 3 blood-derived products (10% HS and 5% ACP: P = .0001, 5% PRP: P = .0002). Cell proliferation at day 9 was significantly increased on stimulation with 10% HS (P = .0001) and 5% PRP (P = .0002) compared with 5% ACP and controls. Meniscus cell pellet cultures showed the formation of a well-structured meniscal ECM with deposition of collagen type I, type II, and proteoglycans on stimulation with 10% HS, whereas 5% PRP or 5% ACP resulted in the formation of an inhomogeneous and more fibrous ECM. Stimulation with 10% HS and 5% ACP showed a significant induction of fibrochondrocyte marker genes such as aggrecan (HS: P = .0002, ACP: P = .0147), cartilage oligomeric matrix protein (HS: P = .0002, ACP: P = .0005), and biglycan (HS: P = .0002, ACP: P = .0003), whereas PRP showed no inducing effect. CONCLUSIONS: Among all tested blood-derived products, only stimulation with HS showed the formation of a meniscal ECM as well as positive cell proliferating and migrating effects in vitro. Regarding a potential biological repair of nonvascular meniscus lesions, our results may point toward the use of HS as a beneficial augment in regenerative meniscus repair approaches. CLINICAL RELEVANCE: Our findings may suggest that HS might be a beneficial augment for meniscus repair.

Repair of retropatellar cartilage defects in the knee with microfracture and a cell-free polymer-based implant.

INTRODUCTION: To analyze magnetic resonance imaging (MRI) at 3T and the clinical outcome in a short-term pilot study after treatment of retropatellar cartilage defects with microfracturing and subsequent covering with the cell-free chondrotissue(®) polyglycolic acid-hyaluronan implant. METHODS: Five consecutive patients after microfracturing and defect coverage with the chondrotissue(®) implant immersed with autologous serum were included. After a mean follow-up of 21 months (range 11-31 months), defect fill and repair tissue quality was assessed by 3-T MRI followed by applying established MRI scoring systems. The patients' situation was assessed using the Knee injury and Osteoarthritis Outcome Score (KOOS) and a patients' satisfaction questionnaire. RESULTS: Magnetic resonance imaging showed good to excellent defect fill with complete integration. The mean MOCART score was 61 (range 50-75) points. The mean Henderson score was 7 (range 6-9) points. All patients showed subchondral bone alterations. The KOOS showed good values in all sub-categories in 4 out of 5 patients and a mean overall score of 73 (range 40-90) points. Two patients rated the outcome as excellent, two as good and one as fair. All patients would have the procedure again and recommend it. CONCLUSIONS: In this small case series, the coverage of symptomatic retropatellar cartilage defects with the chondrotissue(®) implant after microfracturing was safe and feasible with improvement of the patients' situation at short-term follow-up. LEVEL OF EVIDENCE: IV, case series.

Platelet-Rich Plasma Preparation Types Show Impact on Chondrogenic Differentiation, Migration, and Proliferation of Human Subchondral Mesenchymal Progenitor Cells.

PURPOSE: To evaluate the chondrogenic potential of platelet concentrates on human subchondral mesenchymal progenitor cells (MPCs) as assessed by histomorphometric analysis of proteoglycans and type II collagen. Furthermore, the migratory and proliferative effect of platelet concentrates were assessed. METHODS: Platelet-rich plasma (PRP) was prepared using preparation kits (Autologous Conditioned Plasma [ACP] Kit [Arthrex, Naples, FL]; Regen ACR-C Kit [Regen Lab, Le Mont-Sur-Lausanne, Switzerland]; and Dr.PRP Kit [Rmedica, Seoul, Republic of Korea]) by apheresis (PRP-A) and by centrifugation (PRP-C). In contrast to clinical application, freeze-and-thaw cycles were subsequently performed to activate platelets and to prevent medium coagulation by residual fibrinogen in vitro. MPCs were harvested from the cortico-spongious bone of femoral heads. Chondrogenic differentiation of MPCs was induced in high-density pellet cultures and evaluated by histochemical staining of typical cartilage matrix components. Migration of MPCs was assessed using a chemotaxis assay, and proliferation activity was measured by DNA content. RESULTS: MPCs cultured in the presence of 5% ACP, Regen, or Dr.PRP formed fibrous tissue, whereas MPCs stimulated with 5% PRP-A or PRP-C developed compact and dense cartilaginous tissue rich in type II collagen and proteoglycans. All platelet concentrates significantly (ACP, P = .00041; Regen, P = .00029; Dr.PRP, P = .00051; PRP-A, P < .0001; and PRP-C, P < .0001) stimulated migration of MPCs. All platelet concentrates but one (Dr.PRP, P = .63) showed a proliferative effect on MPCs, as shown by significant increases (ACP, P = .027; Regen, P = .0029; PRP-A, P = .00021; and PRP-C, P = .00069) in DNA content. CONCLUSIONS: Platelet concentrates obtained by different preparation methods exhibit different potentials to stimulate chondrogenic differentiation, migration, and proliferation of MPCs. Platelet concentrates obtained by commercially available preparation kits failed to induce chondrogenic differentiation of MPCs, whereas highly standardized PRP preparations did induce such differentiation. These findings suggest differing outcomes with PRP treatment in stem cell-based cartilage repair. CLINICAL RELEVANCE: Our findings may help to explain the variability of results in studies examining the use of PRP clinically.

Enhancing tissue repair in annulus fibrosus defects of the intervertebral disc: analysis of a bio-integrative annulus implant in an in-vivo ovine model.

Annulus fibrosus repair techniques for the intervertebral disc (IVD) address the unsolved problem of reherniation after IVD herniation and might facilitate the development of nucleus pulposus replacement techniques for IVD diseases. This study investigates the suitability of a bio-integrative annulus implant.Standardized box defects were applied to the annulus L3/4 and L4/5 of 16 sheep, followed by randomized insertion of the textile polyglycolic acid/polyvinylidene fluoride annulus implant in one of the defects. Explantation was conducted after 2, 6 and 12 weeks, followed by provocative pressure testing and histological analysis. At 2 weeks' follow-up, all specimens of the control defect group demonstrated uncontained herniated nucleus pulposus tissue in the annulus defects. For the treated specimens, the annulus implant consistently provided an effective barrier for herniating nucleus pulposus tissue, with no implant dislocation at all time-points. After 2 weeks, a homogeneous cell infiltration of the annulus implant was observed, leading to a progressive directional matrix build-up. Repair tissue thickness was significantly stronger with the annulus implant at all follow-ups (p < 0.01). No pronounced foreign body reaction and no difference in the amount of supra-annular scar tissue over the defect sites were observed. The implantation procedure inflicted annulus damage adjacent to the defect. At later time-points, however, no difference in comparison with the control defect group was evident. The investigated biointegrative annulus implant showed promising results with regard to biointegration, enhancement of repair tissue and function as a mechanical barrier in an ovine model.

Fibronectin stimulates migration and proliferation, but not chondrogenic differentiation of human subchondral progenitor cells.

AIMS: To evaluate the impact of human plasma-derived fibronectin (FN) on human subchondral mesenchymal progenitor cells regarding cell migration, proliferation, and chondrogenic differentiation. MATERIALS & METHODS: Human subchondral mesenchymal progenitor cells were analyzed for their migration capacity upon treatment with human plasma-derived FN. Proliferation activity was evaluated by DNA content. For chondrogenesis, cells were cultured in high-density pellet cultures in the presence of FN, TGFß3, and a combination thereof. RESULTS: Treatment of progenitors with FN significantly increased the number of migrating cells and elevated proliferative activity. Histological staining indicated formation of an extracellular matrix with type II collagen. Gene expression analysis gave no evidence for chondrogenic differentiation mediated by FN, but revealed a significant induction of type II collagen expression. CONCLUSION: FN has a potential to recruit human subchondral mesenchymal progenitor cells, possibly supporting proliferation and matrix assembly in cartilage repair procedures using bioactive implants after microfracture treatment.

A 5-year follow-up after cartilage repair in the knee using a platelet-rich plasma-immersed polymer-based implant.

The aim of our study was to analyze the clinical outcome after repair of cartilage defects of the knee with subchondral drilling and resorbable polymer-based implants immersed with autologous platelet-rich plasma (PRP). Fifty-two patients with focal chondral defects were treated with subchondral drilling, followed by covering with a polyglycolic acid - hyaluronan (PGA-HA) implant (chondrotissue®) immersed with autologous PRP. At 5-year follow-up, patients' situation was assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and compared to the pre-operative situation. The KOOS showed clinically meaningful and significant (p < 0.05) improvement in all subcategories compared to baseline. Subgroup analysis showed that there were no differences in the clinical outcome regarding defect size and localization as well as degenerative condition of the knee. Cartilage repair was complete in 20 out of 21 patients at 4-year follow-up as shown by magnetic resonance observation of cartilage repair tissue (MOCART) scoring. Covering of focal cartilage defects with the PGA-HA implant and PRP after bone marrow stimulation leads to a lasting improvement of the patients' situation.

Arthroscopic fixation of cell free polymer-based cartilage implants with a bioinspired polymer surface on the hip joint: a cadaveric pilot study.

This study investigates the adhesion capacity of a polyglycolic acid- (PGA-) hyaluronan scaffold with a structural modification based on a planar polymer (PM) surface in a cadaver cartilage defect model. Two cadaver specimens were used to serially test multiple chondral matrices. In a cadaver hip model, cell free polymer-based cartilage implants with a planar bioinspired PM surface (PGA-PM-scaffolds) were implanted arthroscopically on 10 mm × 15 mm full-thickness femoral hip cartilage lesions. Unprocessed cartilage implants without a bioinspired PM surface were used as control group. The cartilage implants were fixed without and with the use of fibrin glue on femoral hip cartilage defects. After 50 movement cycles and removal of the distraction, a rearthroscopy was performed to assess the outline attachment and integrity of the scaffold. The fixation techniques without and with fibrin fixation showed marginal differences for outline attachment, area coverage, scaffold integrity, and endpoint fixation after 50 cycles. The PGA-PM-scaffolds with fibrin fixation achieved a higher score in terms of the attachment, integrity, and endpoint fixation than the PGA-scaffold on the cartilage defect. Relating to the outline attachment, area coverage, scaffold integrity, and endpoint fixation, the fixation with PGA-PM-scaffolds accomplished significantly better results compared to the PGA-scaffolds (P = 0.03752, P = 0.03078, P = 0.00512, P = 0.00512). PGA-PM-scaffolds demonstrate increased observed initial fixation strength in cadaver femoral head defects relative to PGA-scaffold, particularly when fibrin glue is used for fixation.

Effects of initial boost with TGF-beta 1 and grade of intervertebral disc degeneration on 3D culture of human annulus fibrosus cells.

BACKGROUND: Three-dimensional (3D) culture in porous biomaterials as well as stimulation with growth factors are known to be supportive for intervertebral disc cell differentiation and tissue formation. Unless sophisticated releasing systems are used, however, effective concentrations of growth factors are maintained only for a very limited amount of time in in vivo applications. Therefore, we investigated, if an initial boost with transforming growth factor-beta 1 (TGF-beta 1) is capable to induce a lasting effect of superior cartilaginous differentiation in slightly and severely degenerated human annulus fibrosus (AF) cells. METHODS: Human AF tissue was harvested during surgical treatment of six adult patients with lumbar spinal diseases. Grading of disc degeneration was performed with magnet resonance imaging. AF cells were isolated and expanded in monolayer culture and rearranged three-dimensionally in a porous biomaterial consisting of stepwise absorbable poly-glycolic acid and poly-(lactic-co-glycolic) acid and a supportive fine net of non-absorbable polyvinylidene fluoride. An initial boost of TGF-beta 1 or TGF-beta 1 and hyaluronan was applied and compared with controls. Matrix formation was assessed at days 7 and 21 by (1) histological staining of the typical extracellular matrix molecules proteoglycan and type I and type II collagens and by (2) real-time gene expression analysis of aggrecan, decorin, biglycan, type I, II, III, and X collagens as well as of catabolic matrix metalloproteinases MMP-2 and MMP-13. RESULTS: An initial boost with TGF-beta 1 or TGF-beta 1 and hyaluronan did not enhance the expression of characteristic AF matrix molecules in our 3D culture system. AF cells showed high viability in the progressively degrading biomaterial. Stratification by grade of intervertebral disc degeneration showed that AF cells from both, slightly degenerated, or severely degenerated tissue are capable of significant up-regulations of characteristic matrix molecules in 3D culture. AF cells from severely degenerated tissue, however, displayed significantly lower up-regulations in some matrix molecules such as aggrecan. CONCLUSIONS: We failed to show a supportive effect of an initial boost with TGF-beta 1 in our 3D culture system. This underlines the need for further investigations on growth factor releasing systems.

Initial boost release of transforming growth factor-ß3 and chondrogenesis by freeze-dried bioactive polymer scaffolds.

In cartilage regeneration, bio-activated implants are used in stem and progenitor cell-based microfracture cartilage repair procedures. Our aim was to analyze the chondrogenic potential of freeze-dried resorbable polymer-based polyglycolic acid (PGA) scaffolds bio-activated with transforming growth factor-ß3 (TGFB3) on human subchondral mesenchymal progenitor cells known from microfracture. Progenitor cells derived from femur heads were cultured in the presence of freeze-dried TGFB3 in high-density pellet culture and in freeze-dried TGFB3-PGA scaffolds for chondrogenic differentiation. Progenitor cell cultures in PGA scaffolds as well as pellet cultures with and without continuous application of TGFB3 served as controls. Release studies showed that freeze-dried TGFB3-PGA scaffolds facilitate a rapid, initial boost-like release of 71.5% of TGFB3 in the first 10 h. Gene expression analysis and histology showed induction of typical chondrogenic markers like type II collagen and formation of cartilaginous tissue in TGFB3-PGA scaffolds seeded with subchondral progenitor cells and in pellet cultures stimulated with freeze-dried TGFB3. Chondrogenic differentiation in freeze-dried TGFB3-PGA scaffolds was comparable to cultures receiving TGFB3 continuously, while non-stimulated controls did not show chondrogenesis during prolonged culture for 14 days. These results suggest that bio-activated, freeze-dried TGFB3-PGA scaffolds have chondrogenic potential and are a promising tool for stem cell-mediated cartilage regeneration.

Suitability of porcine chondrocyte micromass culture to model osteoarthritis in vitro.

In vitro tissue models are useful tools for the development of novel therapy strategies in cartilage repair and care. The limited availability of human primary tissue and high costs of animal models hamper preclinical tests of innovative substances and techniques. In this study we tested the potential of porcine chondrocyte micromass cultures to mimic human articular cartilage and essential aspects of osteoarthritis (OA) in vitro. Primary chondrocytes were enzymatically isolated from porcine femoral condyles and were maintained in 96-multiwell format to establish micromass cultures in a high-throughput scale. Recombinant porcine tumor necrosis factor alpha (TNF-α) was used to induce OA-like changes documented on histological (Safranin O, collagen type II staining), biochemical (hydroxyproline assay, dimethylmethylene blue method), and gene expression level (Affymetrix porcine microarray, real time PCR) and were compared with published data from human articular cartilage and human micromass cultures. After 14 days in micromass culture, porcine primary chondrocytes produced ECM rich in proteoglycans and collagens. On gene expression level, significant correlations of detected genes with porcine cartilage (r = 0.90), human cartilage (r = 0.71), and human micromass culture (r = 0.75) were observed including 34 cartilage markers such as COL2A1, COMP, and aggrecan. TNF-α stimulation led to significant proteoglycan (-75%) and collagen depletion (-50%). Comparative expression pattern analysis revealed the involvement of catabolic enzymes (MMP1, -2, -13, ADAM10), chemokines (IL8, CCL2, CXCL2, CXCL12, CCXL14), and genes associated with cell death (TNFSF10, PMAIPI, AHR) and skeletal development (GPNMB, FRZB) including transcription factors (WIF1, DLX5, TWIST1) and growth factors (IGFBP1, -3, TGFB1) consistent with published data from human OA cartilage. Expression of genes related to cartilage ECM formation (COL2A1, COL9A1, COMP, aggrecan) as well as hypertrophic bone formation (COL1A1, COL10A1) was predominantly found decreased. These findings indicating significant parallels between human articular cartilage and the presented porcine micromass model and vice versa confirm the applicability of known cartilage marker and their characteristics in the porcine micromass model. TNF-α treatment enabled the initiation of typical OA reaction patterns in terms of extensive ECM loss, cell death, formation of an inflammatory environment through the induction of genes coding for chemokines and enzymes, and the modulation of genes involved in skeletal development such as growth factors, transcription factors, and cartilage ECM-forming genes. In conclusion, the porcine micromass model represents an alternative tissue platform for the evaluation of innovative substances and techniques for the treatment of OA.

Human platelet-rich plasma induces chondrogenic differentiation of subchondral progenitor cells in polyglycolic acid-hyaluronan scaffolds.

Cartilage repair approaches may be improved by addition of human platelet-rich plasma (PRP) that increases chondrogenic differentiation of mesenchymal stem and progenitor cells. The aim of our study was to evaluate the effect of human PRP on the differentiation of multipotent human subchondral progenitor cells in resorbable polyglycolic acid-hyaluronan (PGA-HA) scaffolds. PGA-HA scaffolds were loaded with subchondral progenitor cells and stimulated with transforming growth factor-beta3 (TGFB3) or 5% PRP, whereas nonstimulated cultures served as controls. Chondrogenic differentiation was evaluated by real-time gene expression analysis of typical chondrogenic marker genes and by immunohistochemical staining of extracellular cartilage matrix molecules such as proteoglycans and collagen type II. TGFB3 and PRP induced the expression of chondrogenic marker genes collagen type II and IX, aggrecan, and cartilage oligomeric matrix protein in subchondral progenitor cells cultured in PGA-HA scaffolds compared with nonstimulated controls. Progenitor cells in PGA-HA scaffolds formed an extracellular matrix rich in proteoglycans and collagen type II on treatment with PRP, but to a lesser extent, than in cultures stimulated with TGFB3. The results suggest that PRP induces chondrogenic differentiation of progenitor cells in PGA-HA scaffolds and may be therefore beneficial in scaffold-assisted cartilage repair approaches involving stem and progenitor cells.

Cartilage repair in the knee with subchondral drilling augmented with a platelet-rich plasma-immersed polymer-based implant.

PURPOSE: The aim of our study was to analyse the clinical and histological outcome after the treatment of focal cartilage defects in non-degenerative and degenerative knees with bone marrow stimulation and subsequent covering with a cell-free resorbable polyglycolic acid-hyaluronan (PGA-HA) implant immersed with autologous platelet-rich plasma (PRP). METHODS: Fifty-two patients (mean age 44 years) with focal chondral defects in radiologically confirmed non-degenerative or degenerative knees were subjected to subchondral drilling arthroscopically. Subsequently, defects were covered with the PGA-HA implant immersed with autologous PRP. At 2-year follow-up, the patients' situation was assessed using the Knee Injury and Osteoarthritis Outcome Score (KOOS) and compared to the pre-operative situation and 3-12-month follow-up. Biopsies (n = 4) were harvested at 18-24 months after implantation and were analysed by histology and collagen type II immune staining. RESULTS: At 1- and 2-year follow-up, the KOOS showed clinically meaningful and significant (p < 0.05) improvement in all subcategories compared to baseline and to 3-month follow-up. There were no differences in KOOS data obtained after 2 years compared to 1 year after the treatment. Histological analysis of the biopsy tissue showed hyaline-like to hyaline cartilage repair tissue that was rich in cells with a chondrocyte morphology, proteoglycans and type II collagen. CONCLUSIONS: Covering of focal cartilage defects with the PGA-HA implant and PRP after bone marrow stimulation improves the patients' situation and has the potential to regenerate hyaline-like cartilage. LEVEL OF EVIDENCE: Case series, Level IV.

Laser-structured bacterial nanocellulose hydrogels support ingrowth and differentiation of chondrocytes and show potential as cartilage implants.

The small size and heterogeneity of the pores in bacterial nanocellulose (BNC) hydrogels limit the ingrowth of cells and their use as tissue-engineered implant materials. The use of placeholders during BNC biosynthesis or post-processing steps such as (touch-free) laser perforation can overcome this limitation. Since three-dimensionally arranged channels may be required for homogeneous and functional seeding, three-dimensional (3-D) laser perforation of never-dried BNC hydrogels was performed. Never-dried BNC hydrogels were produced in different shapes by: (i) the cultivation of Gluconacetobacter xylinus (DSM 14666; synonym Komagataeibacter xylinus) in nutrient medium; (ii) the removal of bacterial residues/media components (0.1M NaOH; 30 min; 100 °C) and repeated washing (deionized water; pH 5.8); (iii) the unidirectional or 3-D laser perforation and cutting (pulsed CO2 Rofin SC × 10 laser; 220 µm channel diameter); and (iv) the final autoclaving (2M NaOH; 121 °C; 20 min) and washing (pyrogen-free water). In comparison to unmodified BNC, unidirectionally perforated--and particularly 3-D-perforated - BNC allowed ingrowth into and movement of vital bovine/human chondrocytes throughout the BNC nanofiber network. Laser perforation caused limited structural modifications (i.e. fiber or globular aggregates), but no chemical modifications, as indicated by Fourier transform infrared spectroscopy, X-ray photoelectron scattering and viability tests. Pre-cultured human chondrocytes seeding the surface/channels of laser-perforated BNC expressed cartilage-specific matrix products, indicating chondrocyte differentiation. 3-D-perforated BNC showed compressive strength comparable to that of unmodified samples. Unidirectionally or 3-D-perforated BNC shows high biocompatibility and provides short diffusion distances for nutrients and extracellular matrix components. Also, the resulting channels support migration into the BNC, matrix production and phenotypic stabilization of chondrocytes. It may thus be suitable for in vivo application, e.g. as a cartilage replacement material.

Is gender influencing the biomechanical results after autologous chondrocyte implantation?

PURPOSE: The influence of gender on the biomechanical outcome after autologous chondrocyte implantation (ACI) including isokinetic muscle strength measurements has not been investigated. The present prospective study was performed to evaluate gender-specific differences in the biomechanical function 48 months after ACI. METHODS: Fifty-two patients (mean age 35.6 ± 8.5 years) that met our inclusion criteria, underwent ACI with Bioseed C(®) and were evaluated with the KOOS score preoperatively, 6, 12 and 48 months after surgery. At final follow-up, 44 out of the 52 patients underwent biomechanical evaluation with isokinetic strength measurements of both knees. All data were evaluated separately for men and women and compared for each time interval using the Mann-Whitney U test. RESULTS: Clinical scores improved significantly over the whole study period (p < 0.05). Male patients demonstrated significantly better scores during the follow-up in the KOOS score (p < 0.05). Isokinetic strength measurements after 48 months revealed a significant strength deficit of the treated knee in all test modes compared to the healthy extremity (p < 0.05). Furthermore, male patients achieved significantly higher strength values compared to female patients (p < 0.05). CONCLUSIONS: ACI is a viable treatment option for full-thickness chondral defects in the knee of both genders. Isokinetic muscle strength measures are significantly worse in women (p < 0.05), but physiological and may play a role for the explanation of gender-specific results after ACI.

Regeneration of nucleus pulposus tissue in an ovine intervertebral disc degeneration model by cell-free resorbable polymer scaffolds.

Degeneration of intervertebral discs (IVDs) occurs frequently and is often associated with lower back pain. Recent treatment options are limited and treat the symptoms rather than regenerate the degenerated disc. Cell-free, freeze-dried resorbable polyglycolic acid (PGA)-hyaluronan implants were used in an ovine IVD degeneration model. The nucleus pulposus of the IVD was partially removed, endoscopically. PGA-hyaluronan implants were immersed in autologous sheep serum and implanted into the disc defect. Animals with nucleotomy only served as controls. The T2-weighted/fat suppression sequence signal intensity index of the operated discs, as assessed by magnetic resonance imaging (MRI), showed that implantation of the PGA-hyaluronan implant improved (p = 0.0066) the MRI signal compared to controls at 6 months after surgery. Histological analysis by haematoxylin and eosin and safranin O staining showed the ingrowth of cells with typical chondrocytic morphology, even cell distribution, and extracellular matrix rich in proteoglycan. Histomorphometric analyses confirmed that the implantation of the PGA-hyaluronan scaffolds improved (p = 0.027) the formation of regenerated tissue after nucleotomy. Disc heights remained stable in discs with nucleotomy only as well as after implantation of the implant. In conclusion, implantation of cell-free polymer-based implants after nucleotomy induces nucleus pulposus tissue regeneration and improves disc water content in the ovine model.

Influence of sex on the outcome of autologous chondrocyte implantation in chondral defects of the knee.

BACKGROUND: Sex-specific outcomes have been reported in anterior cruciate ligament reconstruction as well as in osteoarthrosis progression, but there are currently no related published data on autologous chondrocyte implantation (ACI). The present prospective study was performed to investigate sex-dependent differences in the results after ACI. HYPOTHESIS: The clinical and magnetic resonance imaging (MRI) results after ACI of the knee are influenced by the patient's sex. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: The midterm clinical and MRI results of a cell-based fibrin-polymer graft for the treatment of full-thickness cartilage defects were evaluated preoperatively and 6, 12, and 48 months after surgery in 52 patients (male:female ratio, 25:27; average age, 35.6 years). Depending on the sex and the location of the defects (femoral condyles, n = 32; patellofemoral compartment, n = 20), patients were assigned to 4 different groups. Baseline clinical scores were compared with follow-up data by paired Wilcoxon tests for the Lysholm score and the International Knee Documentation Committee (IKDC) scoring system. Sex-specific differences were evaluated with the Mann-Whitney U test. The MRI evaluation was performed with the Henderson score at final follow-up. RESULTS: Clinical scores improved in all groups over the whole study period (P < .05). Compared with female patients, male patients achieved significantly better results in the Lysholm score at all time intervals and in the IKDC score at 6 and 12 months after surgery (P < .05). In a subgroup analysis, female patients with patellar defects had the worst results in both clinical scores. With the available number of patients, MRI evaluation at 48 months after surgery revealed no significant difference in defect fill between male and female patients (P > .05). The Pearson correlation coefficient between both clinical scores and the MRI parameters of defect fill and cartilage signal was significant (P < .05). CONCLUSION: Autologous chondrocyte implantation is a promising treatment option for full-thickness cartilage defects of male and female knee joints. Female patients with patellar defects have worse prognostic factors.

Polyglycolic acid-hyaluronan scaffolds loaded with bone marrow-derived mesenchymal stem cells show chondrogenic differentiation in vitro and cartilage repair in the rabbit model.

In cartilage repair, scaffold-assisted one-step approaches are used to improve the microfracture (Mfx) technique. Since the number of progenitors in Mfx is low and may further decrease with age, aim of our study was to analyze the chondrogenic potential of freeze-dried polyglycolic acid-hyaluronan (PGA-HA) implants preloaded with mesenchymal stem cells (MSCs) in vitro and in a rabbit articular cartilage defect model. Human bone marrow-derived MSC from iliac crest were cultured in freeze-dried PGA-HA implants for chondrogenic differentiation. In a pilot study, implants were loaded with autologous rabbit MSC and used to cover 5 mm × 6 mm full-thickness femoral articular cartilage defects (n = 4). Untreated defects (n = 3) served as controls. Gene expression analysis and histology showed induction of typical chondrogenic marker genes like type II collagen and formation of hyaline-like cartilaginous tissue in MSC-laden PGA-HA implants. Histological evaluation of rabbit repair tissue formation after 30 and 45 days showed formation of repair tissue, rich in chondrocytic cells and of a hyaline-like appearance. Controls showed no articular resurfacing, tissue repair in the subchondral zone and fibrin formation. These results suggest that MSC-laden PGA-HA scaffolds have chondrogenic potential and are a promising option for stem cell-mediated cartilage regeneration.

A novel in vitro bovine cartilage punch model for assessing the regeneration of focal cartilage defects with biocompatible bacterial nanocellulose.

INTRODUCTION: Current therapies for articular cartilage defects fail to achieve qualitatively sufficient tissue regeneration, possibly because of a mismatch between the speed of cartilage rebuilding and the resorption of degradable implant polymers. The present study focused on the self-healing capacity of resident cartilage cells in conjunction with cell-free and biocompatible (but non-resorbable) bacterial nanocellulose (BNC). This was tested in a novel in vitro bovine cartilage punch model. METHODS: Standardized bovine cartilage discs with a central defect filled with BNC were cultured for up to eight weeks with/without stimulation with transforming growth factor-ß1 (TGF-ß1. Cartilage formation and integrity were analyzed by histology, immunohistochemistry and electron microscopy. Content, release and neosynthesis of the matrix molecules proteoglycan/aggrecan, collagen II and collagen I were also quantified. Finally, gene expression of these molecules was profiled in resident chondrocytes and chondrocytes migrated onto the cartilage surface or the implant material. RESULTS: Non-stimulated and especially TGF-ß1-stimulated cartilage discs displayed a preserved structural and functional integrity of the chondrocytes and surrounding matrix, remained vital in long-term culture (eight weeks) without signs of degeneration and showed substantial synthesis of cartilage-specific molecules at the protein and mRNA level. Whereas mobilization of chondrocytes from the matrix onto the surface of cartilage and implant was pivotal for successful seeding of cell-free BNC, chondrocytes did not immigrate into the central BNC area, possibly due to the relatively small diameter of its pores (2 to 5 µm). Chondrocytes on the BNC surface showed signs of successful redifferentiation over time, including increase of aggrecan/collagen type II mRNA, decrease of collagen type I mRNA and initial deposition of proteoglycan and collagen type II in long-term high-density pellet cultures. Although TGF-ß1 stimulation showed protective effects on matrix integrity, effects on other parameters were limited. CONCLUSIONS: The present bovine cartilage punch model represents a robust, reproducible and highly suitable tool for the long-term culture of cartilage, maintaining matrix integrity and homoeostasis. As an alternative to animal studies, this model may closely reflect early stages of cartilage regeneration, allowing the evaluation of promising biomaterials with/without chondrogenic factors.

Identification of fibronectin as a major factor in human serum to recruit subchondral mesenchymal progenitor cells.

Human serum has the potential for mesenchymal progenitor cell recruitment in repair of articular cartilage lesions. It is unclear which factor(s) in serum mediate this migratory effect. Our goal was to identify cell recruiting factors in human serum fractions obtained by ion exchange chromatography. The recruiting activity of serum fractions on human subchondral mesenchymal progenitor cells was analyzed using 96-well chemotaxis assays. Protein composition of recruiting serum fractions were analyzed by mass spectrometry and showed 58 potential candidates. Fibronectin, gelsolin, lumican, thrombospondin-1 and WNT-9a were identified as key candidates for progenitor cell recruitment. Only human plasma derived and recombinant fibronectin showed significant recruiting activity on progenitors reaching 50-90% of the recruiting activity of normal human serum. Presence of fibronectin in all human serum fractions with recruiting activity was verified by Western blot analysis. This study shows that fibronectin is a key factor in human serum to recruit mesenchymal progenitor cells and might be involved in subchondral mesenchymal progenitor cell migration into cartilage defects after microfracture.