Arthroscopic three dimensional autologous chondrocyte transplantation with navigation-guided cartilage defect size assessment

Introduction

The treatment of large full thickness cartilage defects with matrix guided autologous chondrocyte transplantation shows promising results. However, in many cases an arthrotomy is needed to implant the cell seeded scaffolds. Recently techniques have been developed for arthroscopically guided ACT implantation. Correct defect mapping, to assess size and depth of the chondral lesions, and precise scaffold preparation and fixation are crucial for successful chondrocyte transplantation and remain to be not sufficiently optimized.

Method

In the present study, the geometries of two cartilage defects in cadaver knees were three times assessed, measured and transferred to biodegradable scaffolds with a navigation system by three different executors. The scaffolds were arthroscopically implanted into the cartilage defects.

Results

The cartilage defect assessment was reproducible between all executors for all defect geometries. The implanted scaffolds showed a correct defect filling.

Conclusion

The study showed the feasibility of an arthroscopic implantation of scaffolds for autologous chondrocytes transplantation. Navigation was a useful tool to exactly assess the cartilage defect geometry and allowed a precise transfer of navigated cartilage defect geometries for individualized scaffold preparation. Navigation can help to accomplish and optimize arthroscopically guided chondrocyte transplantations.     

Atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knee: a prospective multicenter clinical trial in Japan

Background

New tissue-engineering technology was developed to create a cartilage-like tissue in a three-dimensional culture using atelocollagen gel. The minimum 2-year followup outcome of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in knees was reported from the single institution. The present multicenter study was conducted to determine clinical and arthroscopic outcomes in patients who underwent atelocollagen-associated autologous chondrocyte implantation for the repair of chondral defects of the knees.

Methods

At six medical institutes in Japan, we prospectively evaluated the clinical and arthroscopic outcomes of transplanting autologous chondrocytes cultured in atelocollagen gel for the treatment of full-thickness defects of cartilage in 27 patients (27 knees) with cartilage lesions on a femoral condyle or on a patellar facet over 24 months.

Results

The Lysholm score significantly increased from 60.0 ± 13.7 points to 89.8 ± 9.5 points (P = 0.001). Concerning the ICRS grade for arthroscopic appearance, 6 knees (24%) were assessed as grade I (normal) and 17 knees (68%) as grade II (nearly normal). There were few adverse features, except for detachment of the graft in two cases.

Conclusions

We concluded that transplanting chondrocytes in a newly formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.     

Cartilage Matrix Formation by Bovine Mesenchymal Stem Cells in Three-dimensional Culture Is Age-dependent

Background

Cartilage degeneration is common in the aged, and aged chondrocytes are inferior to juvenile chondrocytes in producing cartilage-specific extracellular matrix. Mesenchymal stem cells (MSCs) are an alternative cell type that can differentiate toward the chondrocyte phenotype. Aging may influence MSC chondrogenesis but remains less well studied, particularly in the bovine system.

Questions/purposes

The objectives of this study were (1) to confirm age-related changes in bovine articular cartilage, establish how age affects chondrogenesis in cultured pellets for (2) chondrocytes and (3) MSCs, and (4) determine age-related changes in the biochemical and biomechanical development of clinically relevant MSC-seeded hydrogels.

Methods

Native bovine articular cartilage from fetal (n = 3 donors), juvenile (n = 3 donors), and adult (n = 3 donors) animals was analyzed for mechanical and biochemical properties (n = 3–5 per donor). Chondrocyte and MSC pellets (n = 3 donors per age) were cultured for 6 weeks before analysis of biochemical content (n = 3 per donor). Bone marrow-derived MSCs of each age were also cultured within hyaluronic acid hydrogels for 3 weeks and analyzed for matrix deposition and mechanical properties (n = 4 per age).

Results

Articular cartilage mechanical properties and collagen content increased with age. We observed robust matrix accumulation in three-dimensional pellet culture by fetal chondrocytes with diminished collagen-forming capacity in adult chondrocytes. Chondrogenic induction of MSCs was greater in fetal and juvenile cell pellets. Likewise, fetal and juvenile MSCs in hydrogels imparted greater matrix and mechanical properties.

Conclusions

Donor age and biochemical microenvironment were major determinants of both bovine chondrocyte and MSC functional capacity.

Clinical Relevance

In vitro model systems should be evaluated in the context of age-related changes and should be benchmarked against human MSC data.     

Temperature changes and chondrocyte death during drilling in a bovine cartilage model and chondroprotection by modified irrigation solutions

Purpose

Drilling into cartilage/bone is often required for orthopaedic surgery. While drilling into bone has been studied, the response of cartilage has received little attention. We have measured cartilage and drill bit temperatures during drilling and quantified the zone of chondrocyte death (ZCD) around the hole in the presence/absence of irrigation solutions.

Methods

Drilling was performed using a 1.5-mm orthopaedic drill bit applied to bovine metatarsophalangeal joints and temperatures recorded by infrared camera. Osteochondral explants were then incubated with 5-chloromethylfluorescein diacetate (CMFDA) and propidium iodide (PI) to label living/dead chondrocytes respectively. The width of the ZCD was quantified by confocal laser scanning microscopy (CLSM) and image analysis.

Results

Without irrigation, the ZCD following drilling for two seconds was 135?±?15 μm and this increased (>fourfold, P?P?P?265 and 119 °C respectively, whereas the camera saturated at >282 °C during drilling for five seconds. With irrigation, the drill bit temperature was significantly reduced during drilling for two and five seconds (approx. 90 °C) with negligible change in cartilage temperature. Drilling while irrigating with hyperosmotic saline (600 mOsm) reduced (P?P?2+ saline (5 mM).

Conclusions

Reducing temperature during drilling by irrigation markedly suppressed, but did not abolish chondrocyte death. Optimising the irrigation solution by raising osmolarity and reducing Ca²⁺ content significantly reduced chondrocyte death during drilling and may be clinically beneficial.     

Bupivacaine and levobupivacaine induce apoptosis in rat chondrocyte cell cultures at ultra-low doses

Purpose

Osteoarthritis (OA) is characterized by chondrocyte apoptosis and necrosis which play a key role during the progression of OA. Intra-articular administration of bupivacaine is a practical and effective way of postoperative pain control following various joint surgeries. 0.25 % bupivacaine showed to be safe in terms of chondrocyte toxicity. Around 200 nM of bupivacaine was shown to be effective for peripheral nerve block. This study aims to observe the possible cytotoxic effects of bupivacaine and its enantiomer levobupivacaine on chondrocyte cell culture at 7.69, 76.9, and 384.5 μM or at 0.0125, 0.0025, and 0.00025 % concentrations, respectively.

Methods

Chondrocytes were isolated from rat articular cartilage after incubating with collagenase in RPMI-1640 medium. Cells were treated with bupivacaine and levobupivacaine at 7.69, 76.9, and 384.5 μM concentrations for 6, 24, and 48 h. Treated chondrocytes were stained with acridine orange and ethidium bromide and examined under a fluorescence microscope at a 490 nm excitation wavelength for apoptotic changes.

Results

Study results suggest that both bupivacaine and levobupivacaine have dose-dependent chondrocyte toxicity, and this is significantly lesser at 7.69 μM dose. There was no significant difference in terms of chondrocyte apoptosis, (p > 0.05).

Conclusions

Clinicians should be skeptic for the serious long-term side effects of bupivacaine and its analogs, even at ultra-low doses.     

Application of Polyethylene Glycolic Acid Felt with Fibrin Sealant to Prevent Postoperative Pancreatic Fistula in Pancreatic Surgery

Objective

The purpose of this nonrandomized retrospective study was to report our new procedures using polyethylene glycolic acid (PGA) felt with fibrin sealant to prevent severe pancreatic fistula in patients undergoing pancreatic surgery.

Methods

From 2000 to 2008, 54 and 63 patients underwent pancreaticoduodenectomy (PD) and distal pancreatectomy (DP), respectively. Of those patients, we applied PGA felt with fibrin sealant to 18 PD patients and 26 DP patients. In PD patients, the PGA felt was wrapped around the pancreatic suture site, while in DP patients, the PGA felt was wrapped around the predictive division site. The pancreaticojejunostomy site in PD patients and the cut stump in DP patients were coated with fibrin sealant. We compared the occurrence rates for severe postoperative pancreatic fistula (POPF) that occurred after PD or DP both with and without our new procedures.

Results

Before introduction of our procedures, severe POPF developed in 14 of 36 PD patients (39%) and 10 of 37 DP patients (27%). In contrast, after introduction of our procedures, the incidence of POPF was only one in both of 18 PD (6%; P?=?0.016) and 26 DP (4%; P?=?0.017) patients.

Conclusion

In summary, our procedure using PGA felt with fibrin sealant may reduce the risk of severe POPF.     

Combined 3D and hypoxic culture improves cartilage-specific gene expression in human chondrocytes

Background and purpose

In vitro expansion of autologous chondrocytes is an essential part of many clinically used cartilage repair treatments. Native chondrocytes reside in a 3-dimensional (3D) network and are exposed to low levels of oxygen. We compared monolayer culture to combined 3D and hypoxic culture using quantitative gene expression analysis.

Methods

Cartilage biopsies were collected from the intercondylar groove in the distal femur from 12 patients with healthy cartilage. Cells were used for either monolayer or scaffold culture. The scaffolds were clinically available MPEG-PLGA scaffolds (ASEED). After harvesting of cells for baseline investigation, the remainder was divided into 3 groups for incubation in conditions of normoxia (21% oxygen), hypoxia (5% oxygen), or severe hypoxia (1% oxygen). RNA extractions were performed 1, 2, and 6 days after the baseline time point, respectively. Quantitative RT-PCR was performed using assays for RNA encoding collagen types 1 and 2, aggrecan, sox9, ankyrin repeat domain-37, and glyceraldehyde-3-phosphate dehydrogenase relative to 2 hypoxia-stable housekeeping genes.

Results

Sox9, aggrecan, and collagen type 2 RNA expression increased with reduced oxygen. On day 6, the expression of collagen type 2 and aggrecan RNA was higher in 3D culture than in monolayer culture.

Interpretation

Our findings suggest that there was a combined positive effect of 3D culture and hypoxia on cartilage-specific gene expression. The positive effects of 3D culture alone were not detected until day 6, suggesting that seeding of chondrocytes onto a scaffold for matrix-assisted chondrocyte implantation should be performed earlier than 2 days before implantation.Native chondrocytes reside in a 3-dimensional (3D) network of extracellular matrix mainly consisting of collagen type 2 and glycosaminoglycans—primarily aggrecan. The avascular nature of cartilage results in low oxygen tensions. It has been proposed that chondrocytes in the deepest layers of the cartilage are exposed to oxygen tensions of less than 1% (Silver 1975). Culture of chondrocytes in 3D networks such as synthetic biodegradable scaffolds has been shown to be beneficial in terms of retained phenotype in prolonged culture (Takahashi et al. 2007), increased chondrogenic gene expression of dedifferentiated chondrocytes (Barlic et al. 2008), and enhanced proliferation (Lin et al. 2009) compared to monolayer culture. Similarly, hypoxic culture of chondrocytes has been shown to have several substantial effects on cell metabolism. Low oxygen tension induces chondrocyte-specific expression in mesenchymal and embryonic stem cells (Robins et al. 2005, Koay and Athanasiou 2008), stimulates redifferentiation of dedifferentiated and in vitro expanded chondrocytes (Domm et al. 2002, Malda et al. 2004, Murphy and Polak 2004, Martinez et al. 2008), and enhances matrix synthesis after prolonged exposure (Clark et al. 1991, Murphy and Sambanis 2001a, b, Coyle et al. 2009). The effects of hypoxia on chondrocytes are mediated through both sox9-dependent and sox9-independent pathways (Lafont et al. 2008). Although there is a general agreement about several positive effects of hypoxia on chondrocytes, negative effects in bioreactor culture has been described (Obradovic et al. 1999).In treatment of cartilage, both autologous chondrocyte implantation (ACI) and its third-generation counterpart matrix-assisted chondrocyte implantation (MACI) require culture of autologous chondrocytes before reimplantation in the patient (Brittberg et al. 1994). Compared to the native environment, 2 specific factors are altered when chondrocytes are incubated in monolayer culture. The complex 3D structure of cartilage is replaced by a 2-dimensional surface. Secondly, chondrocytes are suddenly exposed to high levels of oxygen. Such culture conditions could alter the chondrogenic phenotype, as prolonged culture of chondrocytes in 21% oxygen has been found to change the phenotype and morphology towards having fibroblastic characteristics (von der Mark et al. 1977).Improvement of the biological response of implanted chondrocytes can be addressed either by selection of “superior” chondrocytes (Dell''Accio et al. 2001, Saris et al. 2008, Katopodi et al. 2009) or by enhancement of culture strategies. We tried to improve traditional chondrocyte culture conditions by combining 3D and hypoxic culture, and we determined the response-specific gene expression in these different culture environments. We hypothesized that culture in an environment resembling the native one would favor the expression of cartilage-specific genes such as sox9, collagen type 2, and aggrecan.     

The oncofetal gene survivin is re-expressed in osteoarthritis and is required for chondrocyte proliferation <Emphasis Type="Italic">in vitro</Emphasis>

Background

Regulation of cell death and cell division are key processes during chondrogenesis and in cartilage homeostasis and pathology. The oncogene survivin is considered to be critical for the coordination of mitosis and maintenance of cell viability during embryonic development and in cancer, and is not detectable in most adult differentiated tissues and cells. We analyzed survivin expression in osteoarthritic cartilage and its function in primary human chondrocytes in vitro.

Methods

Survivin expression was analyzed by immunoblotting and quantitative real-time PCR. The localization was visualized by immunofluorescence. Survivin functions in vitro were investigated by transfection of a specific siRNA.

Results

Survivin was expressed in human osteoarthritic cartilage, but was not detectable in macroscopically and microscopically unaffected cartilage of osteoarthritic knee joints. In primary human chondrocyte cultures, survivin was localized to heterogeneous subcellular compartments. Suppression of survivin resulted in inhibition of cell cycle progression and sensitization toward apoptotic stimuli in vitro.

Conclusions

The present study indicates a role for survivin in osteoarthritic cartilage and human chondrocytes. In vitro experiments indicated its involvement in cellular division and viability. Learning more about the functions of survivin in chondrocyte biology might further help toward understanding and modulating the complex processes of cartilage pathology and regeneration.

     

Severe posttraumatic radiocarpal cartilage damage: first report of autologous chondrocyte implantation

Study design

Case report.

Clinical question

Traumatic articular cartilage defects predispose to secondary osteoarthritis accompanied by impairment or complete loss of function in the corresponding joint. On this account, the timely and correct diagnosis as well as the selection of an appropriate therapy for reconstruction of articular cartilage defects is important.

Methods

A 22-year-old healthy male patient with history of traumatic intra-articular distal radius fracture is presented with in the course detectable 4° cartilage damage in the fovea scaphoidea and into the fovea lunata. For the first time, autologous chondrocyte implantation by the use of an in situ polymerizable albumin–hyaluronic acid gel was performed to restore the articular cartilage.

Results

The assessment 6 months after autologous chondrocyte implantation using the standard DASH questionnaire for upper extremity pointed out an improvement in the patient outcome (DASH score pre-operative: 10.8 and 6 months post-operative: 7.5). The clinical examination also showed an improvement in the range of movement of the wrist without pain. The radiological control investigation (MRI- examination) of the wrist 6 months after implantation also revealed a good integration of the implant.

Conclusion

The case presented here demonstrates the first use of autologous chondrocyte implantation for cartilage reconstruction using an in situ polymerizable albumin–hyaluronic acid gel after traumatic cartilage lesion with 4° cartilage damage of the wrist in a 22-year-old non-smoking, active and healthy man. Initial results are promising in terms of mobility, pain and patient outcome. However, further clinical studies have to be performed with larger number of cases.     

Das bovine Knorpelstanzenmodell

Background

The limited regeneration capacity of hyaline articular cartilage requires detailed studies concerning the tissue integration of cartilage transplants with meaningful but time and/or resource-consuming and in part ethically problematic animal models or, alternatively, with in vitro test systems for implant materials.

Material and methods

The present study describes a regeneration model with bovine cartilage rings (outer Ø 6 mm, central defect Ø 2 mm) for insertion, cultivation and biomechanical or histological testing of cartilage replacement materials (HE and safranin O staining). In this study, resorbable polymers composed of polyglycolic acid (PGA) were analyzed.

Results

Biomechanical testing showed a continuous decrease of the push-out force for the PGA inserts from the cartilage rings, probably due to the resorbability of the material. Histologically, clear immigration of cells into cell-free PGA was observed even after 4 weeks of culture, but in particular after 10 weeks. In addition, storage of proteoglycans was interpreted as an initial sign of the formation of new matrix.

Conclusion

Thus, the new regeneration model is in principle suitable for the testing of biomaterials, but shows limitations in assessing the “lateral bonding” of resorbable materials     

Feasibility of arthroscopic 3-dimensional,purely autologous chondrocyte transplantation for chondral defects of the hip: a case series

Introduction

The purpose of this retrospective study was to describe technical aspects of arthroscopic, purely autologous chondrocyte transplantation of the hip and to report short-term data of the postoperative outcome in a consecutive series of patients.

Materials and methods

We retrospectively analyzed six patients with a full-thickness chondral defect of the hip joint. The defect was treated with an arthroscopically applicable 3-dimensional purely autologous chondrocyte transplant product (chondrosphere^®; co.don^® AG, Berlin, Germany) in a two-step surgical procedure. Patient-administered scores were assessed at baseline (day before transplantation) and at 6 weeks, 3, 6 and 12 months.

Results

Six out of six initially included patients (five males, one female) with a median age of 32.5 years and an average defect size of 3.5 cm² were available for follow-up after a mean of 11.2 months. Five acetabular and one femoral defect were treated. An overall statistically significant improvement was observed for all assessment scores (NHS, mHHS and SF 36).

Conclusion

In this study, we displayed the feasibility and technical aspects of arthroscopic matrix-associated, purely autologous chondrocyte transplantation as a treatment option for full-thickness cartilage defects of the hip. The patient-administered assessment scores demonstrated an increase in activity level and quality of life after a 1-year follow-up.

Level of evidence

Level IV, retrospective study.     

Engineered fetal cartilage: structural and functional analysis in vitro

Background/Purpose: This study was aimed at characterizing the structure and function of engineered fetal cartilage in vitro. Methods: Chondrocytes from ovine specimens of fetal elastic, fetal hyaline, and adult elastic cartilage were expanded in culture and their growth rates determined. Cells were seeded onto synthetic scaffolds, which were then maintained in a bioreactor. Matrix deposition was determined by specific staining and quantitative assays for glycosaminoglycans (GAG), type II collagen (CII), and elastin, as well as compared with native tissue. Statistical analysis was by analysis of variance (ANOVA) and Students' t test, with significance set at P less than .01. Results: Fetal elastic chondrocytes grew significantly faster than all other cell types. All fetal constructs resembled hyaline cartilage, regardless of the cell source. There were significantly higher levels of GAG and CII in fetal versus adult constructs, but no significant difference between fetal constructs from different sources. Unlike their adult counterparts, fetal constructs had GAG and CII levels similar to native tissues. Conclusions: Fetal chondrocytes can be rapidly expanded in culture. Compared with adult constructs, matrix deposition is enhanced in engineered fetal cartilage, which closely resembles native tissue, regardless of the cell source. Engineered fetal cartilage may be a preferable option during surgical reconstruction of select congenital anomalies.     

Inhibition of Gsk3β in cartilage induces osteoarthritic features through activation of the canonical Wnt signaling pathway

Objective

In the past years, the canonical Wnt/β-catenin signaling pathway has emerged as a critical regulator of cartilage development and homeostasis. In this pathway, glycogen synthase kinase-3β (GSK3β) down-regulates transduction of the canonical Wnt signal by promoting degradation of β-catenin. In this study we wanted to further investigate the role of Gsk3β in cartilage maintenance.

Design

Therefore, we have treated chondrocytes ex vivo and in vivo with GIN, a selective GSK3β inhibitor.

Results

In E17.5 fetal mouse metatarsals, GIN treatment resulted in loss of expression of cartilage markers and decreased chondrocyte proliferation from day 1 onward. Late (3 days) effects of GIN included cartilage matrix degradation and increased apoptosis. Prolonged (7 days) GIN treatment resulted in resorption of the metatarsal. These changes were confirmed by microarray analysis showing a decrease in expression of typical chondrocyte markers and induction of expression of proteinases involved in cartilage matrix degradation. An intra-articular injection of GIN in rat knee joints induced nuclear accumulation of β-catenin in chondrocytes 72 h later. Three intra-articular GIN injections with a 2 days interval were associated with surface fibrillation, a decrease in glycosaminoglycan expression and chondrocyte hypocellularity 6 weeks later.

Conclusions

These results suggest that, by down-regulating β-catenin, Gsk3β preserves the chondrocytic phenotype, and is involved in maintenance of the cartilage extracellular matrix. Short term β-catenin up-regulation in cartilage secondary to Gsk3β inhibition may be sufficient to induce osteoarthritis-like features in vivo.     

CaReS® (MACT) versus microfracture in treating symptomatic patellofemoral cartilage defects: a retrospective matched-pair analysis

Background

Treating patellofemoral articular cartilage lesions remains a challenging task in orthopedic surgery. Whereas microfracture and autologous chondrocyte implantation yield good results on femoral condyles, the therapeutic state of the art for treating patellofemoral lesions is yet to be determined. In this study, we compared the CaReS^® technique, which is a matrix-associated autologous chondrocyte implantation technique, to microfracture for treating patellofemoral articular cartilage lesions.

Methods

Between May 2003 and December 2005, 17 patients with an isolated patellofemoral cartilage defect (International Cartilage Repair Society III/IV) were treated with the CaReS^® technique at our department. After adjusting for inclusion and exclusion criteria, ten of these patients could be included in this study; ten patients treated with microfracture were chosen as a matched-pair group. Clinical outcome was evaluated 3 years after surgery by the 36-item Short Form Health Survey Questionnaire (SF-36), International Knee Documentation Committee (IKDC) subjective evaluation of the knee, Lysholm Score, and Cincinnati Modified Rating Scale scores.

Results

Patients treated with CaReS^® had statistically significantly improved IKDC, Lysholm, and Cincinnati scores 36 months after surgery compared with preoperatively. When comparing outcome between groups 36 months after surgery, there was no statistically difference in IKDC, Lysholm, and Cincinnati scores.

Conclusions

This is the first trial comparing the CaReS^® technique and microfracture for treating patellofemoral articular cartilage lesions, and results show that CaReS^® yields comparable results to microfracture. The small number of patients is a limiting factor of the study, leading to results without statistical significance. A multicentric prospective randomized study comparing the two procedures is desirable.     

Migration Inhibitory Factor Enhances Inflammation via CD74 in Cartilage End Plates with Modic Type 1 Changes on MRI

Background

Type 1 Modic changes are characterized by edema, vascularization, and inflammation, which lead to intervertebral disc degeneration. Macrophage migration inhibitory factor (MIF) is a proinflammatory cytokine closely related to the inflammatory cytokines detected in degenerative intervertebral disc tissues. However, the existence and role of MIF and its receptor CD74 in intervertebral disc degeneration have not been elucidated.

Questions/purposes

We asked whether (1) MIF and its receptor CD74 are expressed in cartilage end plates with Type 1 Modic changes, (2) MIF is associated with cartilage end plate degeneration, (3) the MIF antagonist (S, R)-3(4-hydroxyphenyl)-4, 5-dihydro-5-isoxazole acetic acid methyl ester (ISO-1) suppresses MIF-induced inflammatory cytokine release, and (4) inflammatory cytokines are released by cartilage end plate chondrocytes via CD74 by activating the CD74 antibody (CD74Ab).

Methods

We examined MIF and CD74 expression by human cartilage end plate chondrocytes and tissues with Type 1 Modic changes from eight patients using immunocytofluorescence and immunohistochemistry. MIF production by the chondrocytes was assessed by ELISA and PCR. We compared cytokine release by chondrocytes treated with MIF in the presence or absence of exogenous ISO-1 by ELISA. Cytokine release by chondrocytes after treatment with CD74Ab was determined by ELISA.

Results

MIF was expressed in degenerated human cartilage end plate tissues and chondrocytes. Lipopolysaccharide and tumor necrosis factor α (TNF-α) upregulated MIF expression and increased MIF secretion in chondrocytes in a dose-dependent manner. MIF increased the secretion of IL-6, IL-8, and prostaglandin E2 (PGE2) in a dose-dependent manner. ISO-1 reduced the secretion of IL-6, IL-8, and PGE2. CD74Ab activated CD74 and induced release of inflammatory cytokines.

Conclusions

Chondrocytes in cartilage end plate with Type 1 Modic changes express MIF and its receptor CD74. MIF might promote the inflammatory response through CD74. MIF-induced cytokine release appears to be suppressed by ISO-1, and CD74Ab could induce cytokine release.

Clinical Relevance

The MIF/CD74 pathway may represent a crucial target for treating disc degeneration since inhibiting the function of MIF with its antagonist ISO-1 can reduce MIF-induced inflammation and exert potent therapeutic effects.     

Genetic Engineering of Juvenile Human Chondrocytes Improves Scaffold-free Mosaic Neocartilage Grafts

Background

Current cartilage transplantation techniques achieve suboptimal restoration and rely on patient donor cells or living grafts of chondrocytes.

Purpose

We sought to enhance allogeneic grafts by testing mosaics of genetically engineered and naïve juvenile human chondrocytes (jCh).

Methods

We obtained specimens from three humans and performed three experiments (two in vitro, one in vivo). We compared neocartilage with and without (1) supplemented serum-free medium (chondrocyte differentiation medium [CDM]), (2) adenoviral BMP-2 (AdBMP-2) transduction, and (3) varying ratios (0.1–1) of transduced and naïve jCh. We compared (4) healing with mosaic grafts with naïve neocartilage or marrow stimulation in immunosuppressed rats. For each of 10 in vitro treatment groups, we had six replicates for each human, and for each of three in vivo treatment groups, we had four replicates for one human. We scored the histology with the semiquantitative Bern score.

Results

AdBMP-2 and naïve neocartilage growth in CDM were histologically superior (Bern score, 5.2 versus 3.7; 8.0 versus 1.8) and size (8.0 versus 6.1; 7.9 versus 2.2 mg) to standard medium. In CDM, AdBMP-2 decreased viability (76% versus 90%), but increased BMP-2 production (619 ng/mL versus 43 pg/mL). Ten percent and 25% AdBMP-2 transduction had Bern scores of 6.8 and 6.5 and viability of 84% and 83%, respectively. Twenty-five percent mosaic grafts provided better healing histologically than marrow stimulation or naive neocartilage.

Conclusions

Low-level AdBMP-2 and CDM augment neocartilage parameters in vitro and vivo.

Clinical Relevance

Genetic augmentation of jCh and creation of mosaic neocartilage may improve graft viability and articular healing compared with naïve neocartilage.     

Establishment of Parameters for Congenital Thoracic Stenosis: A Study of 700 Postmortem Specimens

Background

Congenital thoracic stenosis (CTS) occurs when the bony anatomy of the canal is smaller than expected in the general population. The diagnosis currently is made based on the clinical impression from subjective radiographic studies, and the normal values for CTS have not been established.

Questions/purposes

We provided a statistical definition for CTS based on objective measurements of thoracic spine specimens and explored parameters that might predict CTS.

Methods

We selected 700 adult skeletal specimens from the Hamann-Todd Collection in the Cleveland Museum of Natural History (Cleveland, OH, USA). We used calipers to measure the sagittal canal diameter (SCD), interpedicle distance (IPD), and pedicle length (PL). At each level, canal area was calculated using a geometric formula, a standard distribution was created, and values two SDs below the mean were considered congenitally stenotic. Corresponding values of SCD and IPD of the stenotic specimens were studied. The values of SCD and IPD predicting CTS with highest sensitivity and specificity were tabulated.

Results

At each level, CTS was defined as: T1, 160?mm²; T2, 135?mm²; T3, 131?mm²; T4, 130?mm², T5, 129?mm², T6, 127?mm²; T7, 127?mm²; T8, 129?mm²; T9, 130?mm²; T10, 132?mm²; T11, 140?mm²; and T12, 173?mm². A SCD less than 15?mm and an IPD less than 18.5?mm were predictive of CTS at each level with sensitivities and specificities of 80% to 100%.

Conclusions

We statistically defined CTS at each level. A SCD less than 15?mm or IPD less than 18.5?mm predicted the presence of CTS at all levels.

Clinical Relevance

In a symptomatic patient, on routine radiologic examination, a physician should suspect stenosis of the thoracic canal if the SCD and IPD are less than 15 and 18.5?mm respectively. As a spinal deformity surgeon, the canal area is especially relevant when considering a possible canal intrusion by implants.     

Osteoarthritis synovial fluid activates pro-inflammatory cytokines in primary human chondrocytes

Purpose

Two of the most common joint diseases are rheumatoid arthritis (RA) and osteoarthritis (OA). Cartilage degradation and erosions are important pathogenetic mechanisms in both joint diseases and have presently gained increasing interest. The aim of the present study was to investigate the effects of the synovial fluid environment of OA patients in comparison with synovial fluids of RA patients on human chondrocytes in vitro.

Methods

Primary human chondrocytes were incubated in synovial fluids gained from patients with OA or RA. The detection of vital cell numbers was determined by histology and by using the Casy Cell Counter System. Cytokine and chemokine secretion was determined by a multiplex suspension array.

Results

Microscopic analysis showed altered cell morphology and cell shrinkage following incubation with synovial fluid of RA patients. Detection of vital cells showed a highly significant decrease of vital chondrocyte when treated with RA synovial fluids in comparison with OA synovial fluids. An active secretion of cytokines such as vascular endothelial growth factor (VEGF) of chondrocytes treated with OA synovial fluids was observed.

Conclusions

Significantly increased levels of various cytokines in synovial fluids of RA, and surprisingly of OA, patients were shown. Activation of pro-inflammatory cytokines of human chondrocytes by synovial fluids of OA patient supports a pro-inflammatory process in the pathogenesis of OA.     

Prevalence and consequences of musculoskeletal symptoms in symphony orchestra musicians vary by gender: a cross-sectional study

Background

Tenascin-C (TN-C) is an extracellular matrix glycoprotein that is involved in tissue injury and repair processes. We analyzed TN-C expression in normal and osteoarthritic (OA) human cartilage, and evaluated its capacity to induce inflammatory and catabolic mediators in chondrocytes in vitro. The effect of TN-C on proteoglycan loss from articular cartilage in culture was also assessed.

Methods

TN-C in culture media, cartilage extracts, and synovial fluid of human and animal joints was quantified using a sandwich ELISA and/or analyzed by Western immunoblotting. mRNA expression of TN-C and aggrecanases were analyzed by Taqman assays. Human and bovine primary chondrocytes and/or explant culture systems were utilized to study TN-C induced inflammatory or catabolic mediators and proteoglycan loss. Total proteoglycan and aggrecanase -generated ARG-aggrecan fragments were quantified in human and rat synovial fluids by ELISA.

Results

TN-C protein and mRNA expression were significantly upregulated in OA cartilage with a concomitant elevation of TN-C levels in the synovial fluid of OA patients. IL-1 enhanced TN-C expression in articular cartilage. Addition of TN-C induced IL-6, PGE₂, and nitrate release and upregulated ADAMTS4 mRNA in cultured primary human and bovine chondrocytes. TN-C treatment resulted in an increased loss of proteoglycan from cartilage explants in culture. A correlation was observed between TN-C and aggrecanase generated ARG-aggrecan fragment levels in the synovial fluid of human OA joints and in the lavage of rat joints that underwent surgical induction of OA.

Conclusions

TN-C expression in the knee cartilage and TN-C levels measured in the synovial fluid are significantly enhanced in OA patients. Our findings suggest that the elevated levels of TN-C could induce inflammatory mediators and promote matrix degradation in OA joints.     

Oligo[poly(ethylene glycol)fumarate] Hydrogel Enhances Osteochondral Repair in Porcine Femoral Condyle Defects

Background

Management of osteochondritis dissecans remains a challenge. Use of oligo[poly(ethylene glycol)fumarate] (OPF) hydrogel scaffold alone has been reported in osteochondral defect repair in small animal models. However, preclinical evaluation of usage of this scaffold alone as a treatment strategy is limited.

Questions/purposes

We therefore (1) determined in vitro pore size and mechanical stiffness of freeze-dried and rehydrated freeze-dried OPF hydrogels, respectively; (2) assessed in vivo gross defect filling percentage and histologic findings in defects implanted with rehydrated freeze-dried hydrogels for 2 and 4 months in a porcine model; (3) analyzed highly magnified histologic sections for different types of cartilage repair tissues, subchondral bone, and scaffold; and (4) assessed neotissue filling percentage, cartilage phenotype, and Wakitani scores.

Methods

We measured pore size of freeze-dried OPF hydrogel scaffolds and mechanical stiffness of fresh and rehydrated forms. Twenty-four osteochondral defects from 12 eight-month-old micropigs were equally divided into scaffold and control (no scaffold) groups. Gross and histologic examination, one-way ANOVA, and one-way Mann-Whitney U test were performed at 2 and 4 months postoperatively.

Results

Pore sizes ranged from 20 to 433 μm in diameter. Rehydrated freeze-dried scaffolds had mechanical stiffness of 1 MPa. The scaffold itself increased percentage of neotissue filling at both 2 and 4 months to 58% and 54%, respectively, with hyaline cartilage making up 39% of neotissue at 4 months.

Conclusions

Rehydrated freeze-dried OPF hydrogel can enhance formation of hyaline-fibrocartilaginous mixed repair tissue of osteochondral defects in a porcine model.

Clinical Relevance

Rehydrated freeze-dried OPF hydrogel alone implanted into cartilage defects is insufficient to generate a homogeneously hyaline cartilage repair tissue, but its spacer effect can be enhanced by other tissue-regenerating mediators.