Cellular therapy and tissue engineering for cartilage repair.

Articular cartilage (AC) has limited capacity for repair. The first attempt to repair cartilage using tissue engineering was reported in 1977. Since then, cell-based interventions have entered clinical practice in orthopaedics, and several tissue engineering approaches to repair cartilage are in the translational pipeline towards clinical application. Classically, these involve a scaffold, substrate or matrix to provide structure, and cells such as chondrocytes or mesenchymal stromal cells to generate the tissue. We discuss the advantages and drawbacks of the use of various cell types, natural and synthetic scaffolds, multiphasic or gradient-based scaffolds, and self-organizing or self-assembling scaffold-free systems, for the engineering of cartilage constructs. Several challenges persist including achieving zonal tissue organization and integration with the surrounding tissue upon implantation. Approaches to improve cartilage thickness, organization and mechanical properties include mechanical stimulation, culture under hypoxic conditions, and stimulation with growth factors or other macromolecules. In addition, advanced technologies such as bioreactors, biosensors and 3D bioprinting are actively being explored. Understanding the underlying mechanisms of action of cell therapy and tissue engineering approaches will help improve and refine therapy development. Finally, we discuss recent studies of the intrinsic cellular and molecular mechanisms of cartilage repair that have identified novel signals and targets and are inspiring the development of molecular therapies to enhance the recruitment and cartilage reparative activity of joint-resident stem and progenitor cells. A one-fits-all solution is unrealistic, and identifying patients who will respond to a specific targeted treatment will be critical.

YAP/TAZ regulates the expression of proteoglycan 4 and tenascin C in superficial-zone chondrocytes.

The roles of cell division control protein 42 homologue (CDC42) and actin polymerisation in regulating the phenotype of superficial-zone chondrocytes (SZCs) have been demonstrated in vitro; however, the signalling pathway(s) downstream have yet to be fully elucidated. The study hypothesis was that Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) act downstream to regulate proteoglycan 4 (PRG4) and tenascin C (TNC). Bovine SZCs grown in monolayer were treated with ML141 (CDC42 inhibitor) or the actin depolymerising agents, latrunculin B and cytochalasin D, to determine the effect on YAP/TAZ. Verteporfin (YAP/TAZ inhibitor) and YAP/TAZ siRNA-mediated knockdown were used to determine their role in regulating PRG4 and TNC. ML141 treatment reduced total YAP/TAZ protein, nuclear TAZ levels and the YAP/TAZ target gene, connective tissue growth factor (CTGF) mRNA levels. Latrunculin B decreased nuclear TAZ, while cytochalasin D treatment trended towards increased nuclear TAZ (p = 0.06), correlating with decreased and increased CTGF mRNA levels, respectively. Verteporfin treatment decreased PRG4 and TNC expression, with no effect on actin polymerisation. siRNA-mediated knockdown of YAP/TAZ revealed that PRG4 was regulated by YAP/TAZ while TNC was regulated by TAZ only. As cytochalasin D can activate myocardin-related transcription factor-A (MRTF-A), siRNA-mediated knockdown was performed to determine the role of MRTF-A in regulating YAP/TAZ. Although nuclear TAZ decreased, no significant changes in total protein levels were observed. Findings suggested that CDC42 and actin polymerisation regulated SZCs through multiple actin-regulated pathways. Understanding the regulation of these chondroprotective molecules may have important implications for prevention/treatment of osteoarthritis.

Processing and properties of Na-doped porous calcium polyphosphates - Mechanical properties and in vitro degradation characteristics.

Porous calcium polyphosphate (CPP) is being investigated for use as a biodegradable bone substitute and for repair of osteochondral defects. The necessary requirements for these applications, particularly in load-bearing sites, include sufficient strength to withstand functional forces prior to bone ingrowth and substitution of the initial porous CPP template with new bone and cartilage (for osteochondral implants) in a timely and efficacious manner. The present study explored the effects of Na+ doping and processing to form porous structures of both higher strength and faster degradation than previously reported for 'pure' (non-doped) CPP structures of similar geometry. Compressive and tensile strengths were determined before and after 30-day in vitro degradation (PBS, pH 7.1 at 37 °C) and degradation rates assessed. Scanning electron microscopy (SEM), x-ray diffraction (XRD) and solid state nuclear magnetic resonance (31P SS NMR) were used to evaluate 'pure' and Na-doped CPP samples before and after degradation. The results indicated that the different processing protocols required to prepare samples of similar volume % porosity (a 2-step procedure with a Step-1 sintering temperatures equal to 575 °C being used with the Na-doped samples versus a 585 °C Step-1 treatment for 'pure' CPP) resulted in an approximate 1.5- to 2-fold increase in strength (tensile & compressive respectively) and 2-fold increase in degradation rate of Na-doped CPP compared with 'pure' CPP. This difference was attributed to the different Step-1 sintering temperatures used for sample processing.

Engineering of hyaline cartilage with a calcified zone using bone marrow stromal cells.

OBJECTIVE: In healthy joints, a zone of calcified cartilage (ZCC) provides the mechanical integration between articular cartilage and subchondral bone. Recapitulation of this architectural feature should serve to resist the constant shear force from the movement of the joint and prevent the delamination of tissue-engineered cartilage. Previous approaches to create the ZCC at the cartilage-substrate interface have relied on strategic use of exogenous scaffolds and adhesives, which are susceptible to failure by degradation and wear. In contrast, we report a successful scaffold-free engineering of ZCC to integrate tissue-engineered cartilage and a porous biodegradable bone substitute, using sheep bone marrow stromal cells (BMSCs) as the cell source for both cartilaginous zones. DESIGN: BMSCs were predifferentiated to chondrocytes, harvested and then grown on a porous calcium polyphosphate substrate in the presence of triiodothyronine (T3). T3 was withdrawn, and additional predifferentiated chondrocytes were placed on top of the construct and grown for 21 days. RESULTS: This protocol yielded two distinct zones: hyaline cartilage that accumulated proteoglycans and collagen type II, and calcified cartilage adjacent to the substrate that additionally accumulated mineral and collagen type X. Constructs with the calcified interface had comparable compressive strength to native sheep osteochondral tissue and higher interfacial shear strength compared to control without a calcified zone. CONCLUSION: This protocol improves on the existing scaffold-free approaches to cartilage tissue engineering by incorporating a calcified zone. Since this protocol employs no xenogeneic material, it will be appropriate for use in preclinical large-animal studies.

A systematic review of active treatment options in patients with desmoid tumours.

INTRODUCTION: We conducted a systematic review to determine the optimal treatment options in patients with desmoid tumours who have declined observational management. METHODS: A search was conducted of the medline and embase databases (1990 to September 2012), the Cochrane Library, and relevant guideline Web sites and conference materials. RESULTS: One systematic review and forty-six studies met the preplanned study selection criteria; data from twenty-eight articles were extracted and analyzed. For local control, three studies reported a statistically significant difference in favour of surgery plus radiotherapy (rt) compared with surgery alone, and one study did not; two studies reported the lack of a statistical difference between surgery plus rt and rt alone in maintaining local control. Multivariate risk factors for local recurrence included positive surgical margins and young patient age. Single-agent imatinib led to a progression-free survival rate of 55% at 2 years and 58% at 3 years. Methotrexate plus vinblastine led to a progression-free survival rate of 67% at 10 years. Significant toxicities were reported for all treatment modalities, including surgical morbidity, and rt- and chemotherapy-related toxicities. CONCLUSIONS: In patients who have declined observational management, the local control rate was higher with surgery plus rt than with surgery alone. However, the additional rt-related complications should be considered in treatment decision-making. Surgery, rt, and systemic therapy are all reasonable treatment options for patients with desmoid tumours.

Treatment and follow-up strategies in desmoid tumours: a practice guideline.

OBJECTIVES: We set out to determine the optimal treatment options-surgery, radiation therapy (rt), systemic therapy, or any combinations thereof-for patients with desmoid tumours once the decision to undergo active treatment has been made (that is, monitoring and observation have been determined to be inadequate).provide clinical-expert consensus opinions on follow-up strategies in patients with desmoid tumours after primary interventional management. METHODS: This guideline was developed by Cancer Care Ontario's Program in Evidence-Based Care and the Sarcoma Disease Site Group. The medline, embase, and Cochrane Library databases, main guideline Web sites, and abstracts of relevant annual meetings (1990 to September 2012) were searched. Internal and external reviews were conducted, with final approval by the Program in Evidence-Based Care and the Sarcoma Disease Site Group. RECOMMENDATIONS TREATMENTS: Surgery with or without rt can be a reasonable treatment option for patients with desmoid tumours whose surgical morbidity is deemed to be low.The decision about whether rt should be offered in conjunction with surgery should be made by clinicians and patients after weighing the potential benefit of improved local control against the potential harms and toxicity associated with rt.Depending on individual patient preferences, systemic therapy alone or rt alone might also be reasonable treatment options, regardless of whether the desmoid umours are deemed to be resectable. RECOMMENDATIONS FOLLOW-UP STRATEGIES: Undergo evaluation for rehabilitation (occupational therapy or physical therapy, or both).Continue with rehabilitation until maximal function is achieved.Undergo history and physical examinations with appropriate imaging every 3-6 months for 2-3 years, and then annually.

4-Pyridyl-substituted azacycloalkanes with multiple donor sites prepared via a solvent-free, one-pot method for use in electrocatalysts.

We report the first, solvent-free synthesis of 4-pyridyl tacn, cyclen, and cyclam derivatives as new design elements to prepare electrocatalysts, for example, by the differential metal complexation of 4-py(n)cyclen (n = 2, 4).

The clinical status of cartilage tissue regeneration in humans.

PURPOSE: To provide a comprehensive overview of the basic science and clinical evidence behind cartilage regeneration techniques as they relate to surgical management of chondral lesions in humans. METHODS: A descriptive review of current literature. RESULTS: Articular cartilage defects are common in orthopedic practice, with current treatments yielding acceptable short-term but inconsistent long-term results. Tissue engineering techniques are being employed with aims of repopulating a cartilage defect with hyaline cartilage containing living chondrocytes with hopes of improving clinical outcomes. Cartilage tissue engineering broadly involves the use of three components: cell source, biomaterial/membranes, and/or growth stimulators, either alone or in any combination. Tissue engineering principles are currently being applied to clinical medicine in the form of autologous chondrocyte implantation (ACI) or similar techniques. Despite refinements in technique, current literature fails to support a clinical benefit of ACI over older techniques such as microfracture except perhaps for larger (>4 cm) lesions. Modern ACI techniques may be associated with lower operative revision rates. The notion that ACI-like procedures produce hyaline-like cartilage in humans remains unsupported by high-quality clinical research. CONCLUSIONS: Many of the advancements in tissue engineering have yet to be applied in a clinical setting. While basic science has refined orthopedic management of chondral lesions, available evidence does not conclude the superiority of modern tissue engineering methods over other techniques in improving clinical symptoms or restoring native joint mechanics. It is hoped further research will optimize ease of cell harvest and growth, enhanced cartilage production, and improve cost-effectiveness of medical intervention.

Surgical margins and handling of soft-tissue sarcoma in extremities: a clinical practice guideline.

QUESTIONS: In limb salvage surgery for extremity soft-tissue sarcoma (sts), what is an adequate surgical margin?What is the appropriate number of samples to take from the margins of a surgical resection specimen?What is the appropriate handling of surgical resection specimens? BACKGROUND: Surgery is the primary treatment for extremity sts. The combination of radiotherapy with surgery allows for limb salvage by using radiation to biologically "sterilize" microscopic extensions of tumour and to spare neurovascular and osseous structures. Adjuvant chemotherapy in sts-except for rhabdomyosarcoma and Ewing sarcoma-continues to be controversial. METHODS: The medline and embase databases (1975 to June 2011) and the Cochrane Library were searched for pertinent studies. The Web sites of the main guideline organizations and the American Society of Clinical Oncology conference proceedings (2007-2010) were also searched. RESULTS AND CONCLUSIONS: Thirty-three papers, including four guidelines, one protocol, and one abstract, were eligible for inclusion. The data suggest that patients with clear margins have a better prognosis, but no prospective studies have indicated how wide margins should be. In limb-salvage surgery for extremity sts, the procedure should be planned to achieve a clear margin. However, to preserve functionality, surgery may result in a very close (<1 cm) or even microscopically positive margin. In this circumstance, the use of preoperative or postoperative radiation should be considered. No studies described the optimal number of tissue sections required to assess adequacy of excision nor the appropriate handling of surgical resection specimens. The Sarcoma Disease Site Group made its recommendations based on expert opinion and consensus.

Primary cilia attenuate hedgehog signalling in neoplastic chondrocytes.

Primary cilia can act as either a negative or positive regulator of the hedgehog (Hh) signaling pathway. Many cartilage tumors are characterized by abnormal activation of the Hh pathway. Here, we report that the presence of primary cilia occurs at a low frequency (12.4%) in neoplastic chondrocytes from malignant human chondrosarcomas, compared with chondrocytes from normal articular cartilage (67.7%). To determine the function of primary cilia in cartilaginous neoplasia, we studied benign cartilage tumors that are formed in mice by chondrocyte-specific overexpression of Gli2, a downstream transcriptional activator of the Hh pathway. Col2A1-Gli2 mice were crossed with Ift88+/- mice, which display a partial loss of ciliogenesis. Surprisingly, cartilage tumors developed in Ift88+/- mice that were phenotypically similar to those that arise in Col2A1-Gli2 mice. Further activation of the Hh pathway was observed in Col2A1-Gli2; Ift88+/- mice compared with either Col2A1-Gli2 or Ift88+/- mice, which was associated with an increased incidence of cartilage tumors. Chondrosarcomas were established in explant cultures, and treated with choral hydrate, which disrupts the functional primary cilia. Thus, treatment resulted in hyperactivity of the Hh signaling pathway, as well as cellular changes that could promote tumor growth. Primary cilia functions to inhibit Hh signaling in neoplastic chondrocytes. The activation of Hh signaling is sufficient to induce benign cartilage tumors without another oncogenic initiating event. Moreover, as primary cilia suppress Hh pathway activation in chondrosarcoma, cellular mechanisms inhibiting proper cilia function may be important in maintaining the neoplastic phenotype.

Characterization of the annulus fibrosus-vertebral body interface: identification of new structural features.

Current surgical treatments for degenerative intervertebral disc disease do not restore full normal spinal movement. Tissue engineering a functional disc replacement may be one way to circumvent this limitation, but will require an integration of the different tissues making up the disc for this approach to be successful. Hence, an in-depth characterization of the native tissue interfaces, including annulus insertion into bone is necessary, as knowledge of this interface is limited. The objective of this study was to characterize the annulus fibrosus-vertebral bone (AF-VB) interface in immature (6-9 months old) and mature (18-24 months old) bovine discs, as well as to define these structures for normal adult human (22 and 45 years old) discs. Histological assessment showed that collagen fibers in the inner annulus, which are predominantly type II collagen, all appear to insert into the mineralized endplate zone. In contrast, some of the collagen fibers of the outer annulus, predominantly type I collagen, insert into this endplate, while other fibers curve laterally, at an ∼ 90° angle, to the outer aspect of the bone, and merge with the periosteum. This is seen in both human and bovine discs. Where the AF inserts into the calcified zone of the AF-VB interface, it passes through a chondroid region, rich in type II collagen and proteoglycans. Annulus cells (elongated cells that are not surrounded by proteoglycans) are present at this interface. This cartilage zone is evident in both human and bovine discs. Type X collagen and alkaline phosphatase are localized to the interface region. Age-associated differences in bovine spines are observed when examining the interface thickness and the matrix composition of the cartilaginous endplate, as well as the thickness of the mineralized endplate. These findings will assist with the design of the AF-VB interface in the tissue engineered disc.

Enhanced levels of non-enzymatic glycation and pentosidine crosslinking in spontaneous osteoarthritis progression.

OBJECTIVE: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content in cartilage accelerates the progression of spontaneous osteoarthritis (OA) in the Hartley guinea pig (HGP) model. METHODS: Twenty-eight male, 3-month-old HGPs were used. Eight were left untreated as a baseline control group and sacrificed at 3 months of age (n = 4) and 9 months of age (n = 4; age-matched controls). The other 20 HGPs received intra-articular knee injections in the right knee whereas the left knees acted as contra-lateral non-injected controls. Injections consisted of 100 µl phosphate buffered saline (PBS; n = 10) or PBS+2.0 M D-(-)-Ribose (n = 10). Injections were given once weekly for 24 weeks. At the end of the treatment period, the tibiae were fixed with formalin, scanned with microCT for sub-chondral bone mineral density, and then histological slides were prepared, stained with Safranin-O with Fast Green counter stain and scored using the OARSI-HISTOgp scheme. Cartilage pentosidine (established biomarker for AGEs) content, collagen content (% dry mass), glucosaminoglycan GAG-to-collagen ratio (µg/µg), GAG-to-DNA ratio and DNA-to-collagen ratio were measured. RESULTS: Pentosidine content increased greatly due to PBS + Ribose injection (P < 0.0001) and reached levels found in cartilage from 80-year-old humans. Surprisingly, mean OARSI-HISTOgp scores for both the injected and contra-lateral controls in the PBS + Ribose group were not detectably different, nor were they different from the mean score for the age-matched control group. CONCLUSION: AGEs accumulation due to intra-articular ribose-containing injections in the HGP model of spontaneous knee OA did not enhance disease progression.

Clinical features, treatment, and outcome in 102 adult and pediatric patients with localized high-grade synovial sarcoma.

Background. There remains controversy on the routine use of chemotherapy in localized SS. Methods. The records of 87 adult (AP) and 15 pediatric (PP) patients with localized SS diagnosed between 1986 and 2007 at 2 centres in Toronto were reviewed. Results. Median age for AP and PP was 37.6 (range 15-76) and 14 (range 0.4-18) years, respectively. 65 (64%) patients had large tumours (>5 cm). All patients underwent en bloc surgical resection resulting in 94 (92.2%) negative and 8 (7.8%) microscopically positive surgical margins. 72 (82.8%) AP and 8 (53%) PP received radiotherapy. Chemotherapy was administered to 12 (13.8%) AP and 13 (87%) PP. 10 AP and 5 PP were evaluable for response to neoadjuvant chemotherapy, with response rate of 10% and 40%, respectively. 5-year EFS and OS was 69.3 ± 4.8% and 80.3 ± 4.3%, respectively, and was similar for AP and PP, In patients with tumors >5 cm, in whom chemotherapy might be considered most appropriate, relapse occurred in 9/19 (47%) with chemotherapy, compared to 17/46 (37%) In those without. Conclusions. Patients with localized SS have a good chance of cure with surgery and RT. Evidence for a well-defined role of chemotherapy to improve survival In localized SS remains elusive.

The efficacy of chemical adjuvants on giant-cell tumour of bone. An in vitro study.

Various chemicals are commonly used as adjuvant treatment to surgery for giant-cell tumour (GCT) of bone. The comparative effect of these solutions on the cells of GCT is not known. In this study we evaluated the cytotoxic effect of sterile water, 95% ethanol, 5% phenol, 3% hydrogen peroxide (H(2)O(2)) and 50% zinc chloride (ZnCI(2)) on GCT monolayer tumour cultures which were established from six patients. The DNA content, the metabolic activity and the viability of the cultured samples of tumour cells were assessed at various times up to 120 hours after their exposure to these solutions. Equal cytotoxicity to the GCT monolayer culture was observed for 95% ethanol, 5% phenol, 3% H(2)O(2) and 50% ZnCI(2). The treated samples showed significant reductions in DNA content and metabolic activity 24 hours after treatment and this was sustained for up to 120 hours. The samples treated with sterile water showed an initial decline in DNA content and viability 24 hours after treatment, but the surviving cells were viable and had proliferated. No multinucleated cell formation was seen in these cultures. These results suggest that the use of chemical adjuvants other than water could help improve local control in the treatment of GCT of bone.

Blindness prevalence and cataract surgical coverage in Lumbini Zone and Chetwan District of Nepal.

AIM: To determine the prevalence of blindness, visual impairment and the cataract surgical coverage for people aged 50 years and older in the Lumbini Zone and the Chitwan District (Narayani Zone) of Nepal. METHODS: A population-based cross-sectional study in 2006 selected subjects aged 50 years and older through a random multistage cluster sampling and door-to-door enumeration. Ophthalmic examination included visual-acuity assessment and refraction, and anterior and posterior segment examination of the eyes carried out by a trained ophthalmologist and two ophthalmic assistants at centralised locations. RESULTS: The survey examined 5138 of 5196 persons enumerated (response rate of 86.8%). The mean age of the subjects was 61 (SD 9.2) years, and 2701 (52.6%) subjects were women. The age-sex-adjusted prevalence of blindness (best presenting vision <6/60) and visual impairment (better-eye presenting visual acuity of <6/18 to > or = 6/60) were 4.6% (95% CI 3.4 to 5.8) and 18.9% (95% CI 16.4 to 21.4), respectively. Blindness was significantly lower in the hill (3.3%) compared with the plain (5.8%) regions (OR 0.6; 95% CI 0.4 to 0.9). The primary causes for blind eyes were cataract (n = 228, 48.1%), refractive error (n = 149, 31.4%), retinal disorders (n = 19, 4.0%) and corneal opacity (n = 18, 3.8%). The overall cataract surgical coverage was 66.6%. Cataract surgical coverage was not significantly associated with age, sex, literacy or District. CONCLUSION: Although the prevalence of blindness and visual impairment is lower than 10 years ago, particularly among women, correctable blindness due to cataract and refractive error (79.5% of blind people) remains a significant population health problem in Lumbini Zone and Chitwan District.

Causes of vision impairment and assessment of need for low vision services for students of blind schools in Nepal.

BACKGROUND: The present study is first of its kind to evaluate causes of visual impairment of blind students in Nepal and assess their need for low vision rehabilitation services. AIM: To evaluate causes of vision impairment of students enrolled in blind schools in Nepal and assess the need for low vision rehabilitation services in these students. MATERIALS AND METHODS: A survey was conducted in 12 blind schools in Nepal, which were registered with Nepal Association for Welfare of Blindness (NAWB).It was conducted by a team of an ophthalmologist and an optometrist, by using standard eye examination protocols of the World Health Organization Prevention of Blindness Program (WHO/PBL). RESULTS: Of the 345 students enrolled in 12 schools, 285 students were examined (response rate of 82.61%). The students were in the 5 - 29 years age group. Nearly three-fourth of the children had become blind within one year of age and 52.3% visually impaired at birth and 20.7% developed vision impairment within one year of age. After refraction, 26 students (9.12%) had mild visual impairment, 21 students (7.37%) had severe visual impairment and 238 students (83.51%) were blind. The main cause of vision impairment was found to be corneal 35.79% and retina diseases, mainly dystrophy, 20.35% followed by problems with the whole globe, lens and optic nerve, accounting for 13.33%, 12.63% and 12.98% respectively. The major etiological factors were those of childhood such as Vitamin A deficiency, measles and similar causes (42.11%) followed by hereditary causes (25.26%). Of the total students examined, 48.07% were visually impaired due to preventable causes and 16.14% treatable aggregating to 64.21% of avoidable blindness. Fifty seven (28.22%) students could read smaller than 2 M print size after low vision assessment for near and 33(15.78%) students benefited with telescopic trial for distance low vision. CONCLUSION: In Nepal, renewed focus on providing best possible quality of life for visually impaired children by proper low vision assessment and eye health education focusing on, general public and community health workers, with governmental and institutional support is required to achieve Vision 2020 objectives to decrease childhood blindness.

AP-1 DNA binding activity regulates the cartilage tissue remodeling process following cyclic compression in vitro.

Generating bioengineered cartilage yields tissue with physical qualities inferior to that of native tissue. Application of cyclic compression (30 min, 1 kPa, 1 Hz) to cartilage cells (chondrocytes) seeded on calcium polyphosphate substrates significantly increases the accumulation of collagens and proteoglycans by 24 hours, thus improving the tissue generated. The mechanism for this increase is not fully known but seems to follow a remodeling pathway of sequential catabolic and anabolic changes. The initial catabolic event involves increased transcription of matrix metalloproteinase (MMP)-3 and MMP-13 two hours after the end of cyclic compression. As MMP-3 and MMP-13 promoters contain activating protein-1 (AP-1) DNA binding sites, we investigated the effect of inhibiting DNA binding through the use of modified decoy oligodeoxynucleotides (ODN). Mechanical stimulation in the presence of the ODN blocked AP-1 DNA binding as detected by electrophoretic mobility shift assay and prevented the increased transcription of MMP-3 and MMP-13. As well the increased accumulation of collagens and proteoglycans by 24 hours in mechanically stimulated samples was prevented. The data suggests that the mechano-induction of MMP-3 and MMP-13 may be regulated at the AP-1 DNA binding site and that upregulation of these metalloproteases is a necessary component of the matrix remodeling initiated by cyclic compression.

An in vitro tissue model to study the effect of age on nucleus pulposus cells.

Differentiation between age (physiological) and disease-induced changes in the nucleus pulposus will facilitate our understanding of the mechanism(s) leading to the development of degenerative disc disease. The aim of this study was to develop an in vitro model that would allow the study of age-induced alterations of cell function in nucleus pulposus. Nucleus pulposus (NP) cells were isolated from intervertebral discs obtained from either calves (<9 months) or cows (>18 months). The cells were placed in culture and grown for 19 days. Although nucleus pulposus tissue was formed by the cells of the two different ages the more mature (older) cells formed less tissue as determined histologically by light microscopy. This was confirmed biochemically as the wet weight and proteoglycan content of the tissue formed by the older cells were significantly less than that of the younger tissue. The older cells accumulated less proteoglycans as determined by quantifying radioisotope incorporation. The older cells showed lower constitutive gene expression of collagen type II and aggrecan whereas collagen type I and link protein levels were similar to those of the younger cells. Metalloprotease (MMP) 13 gene and protein expression increased with age. There was no change in the levels of gene expression of MMP 2 and TIMP 1, 2, or 3 with age. Cells obtained from NP tissue harvested from younger or mature animals showed both genotypic and phenotypic differences in vitro that resulted in the inability of the older cells to reconstitute their extracellular matrix to the same extent as the younger cells. This suggests that this in vitro NP tissue model will be suitable to determine the mechanism(s) regulating age-induced changes.

Membrane type-1 matrix metalloproteinase is induced following cyclic compression of in vitro grown bovine chondrocytes.

OBJECTIVE: To determine if membrane type-1 matrix metalloproteinase (MT1-MMP) will respond to cyclic compression of chondrocytes grown in vitro and the regulatory mechanisms underlying this response. METHODS: Cyclic compression (30min, 1kPa, 1Hz) was applied to bovine chondrocytes (6-9-month-old animals) grown on top of a biodegradable substrate within 3 days of initiating culture. Luciferase assays using bovine articular chondrocytes were undertaken to demonstrate the mechanosensitivity of MT1-MMP. Semi-quantitative reverse-transcription polymerase chain reaction (RT-PCR) and western blot analysis were used to establish the time course of gene and protein upregulation in response to cyclic compression. The regulation of MT1-MMP was assessed by electrophoretic mobility shift assays, RT-PCR and western blot analysis. As well, an MT1-MMP decoy oligonucleotide and an extracellular signal-regulated kinase 1/2 (ERK1/2) pharmacological inhibitor were utilized to further characterize MT1-MMP regulation. RESULTS: After cyclic compression, MT1-MMP showed a rapid and transient increase in gene expression. Elevated protein levels were detected within 2h of stimulation which returned to baseline by 6h. During cyclic compression, phosphorylation of the mitogen activated protein kinase ERK1/2 increased significantly. This was followed by increased gene and protein expression of the transcription factor; early growth factor-1 (Egr-1) and Egr-1 binding to the MT1-MMP promoter. Blocking Egr-1 DNA binding with a decoy MT1-MMP oligonucleotide, downregulated MT1-MMP gene expression. The ERK1/2 inhibitor U0126 also reduced Egr-1 DNA binding activity to MT1-MMP promoter sequences and subsequent transcription of MT1-MMP. CONCLUSIONS: These data suggest that cyclic compression of chondrocytes in vitro upregulates MT1-MMP via ERK1/2 dependent activation of Egr-1 binding. Delineation of the regulatory pathways activated by mechanical stimulation will further our understating of the mechanisms influencing tissue remodeling.

Formation of biphasic constructs containing cartilage with a calcified zone interface.

The zone of calcified cartilage is the mineralized region of articular cartilage that anchors the hyaline cartilage to the subchondral bone and serves to disperse mechanical forces across this interface. In an attempt to mimic this zonal organization, we have developed the methodology to form biphasic constructs composed of cartilaginous tissue anchored to the top surface of a bone substitute (porous calcium polyphosphate, CPP) with a calcified interface. To accomplish this, chondrocytes were selectively isolated from the deep zone of bovine articular cartilage, placed on top of the CPP substrate, and grown in the presence of beta-glycerophosphate (10 mM, beta-GP). By 8 weeks, cartilage tissue had formed with two zones: a calcified region adjacent to the CPP substrate and a hyaline-like zone above. Little or no mineralization occurred in the absence of beta-GP. The mineral that formed in vitro was identified as hydroxyapatite, similar in composition and crystal size to that found in vivo. The tissue stiffness was seven times greater, and the interfacial shear properties at the cartilage-CPP interface were at least two times greater in the presence of this mineralized zone within the in vitro-formed cartilage than in tissue lacking a mineral zone. In conclusion, developing a biphasic construct with a calcified zone at the tissue-biomaterial interface resulted in significantly better cartilage load-bearing (compressive) properties and interfacial shear strength, emphasizing the importance of the presence of a mineralized zone in bioengineered cartilage. Because failure due to shear occurred at the cartilage-CPP interface instead of the tidemark, as occurs with osteochondral tissue, further study is required to optimize this system so that it more closely mimics the native tissue.