Neurotropin® ameliorates chronic pain associated with scar formation in a mouse model: A gene expression analysis of the inflammatory response.

Background: Scar formation after trauma and surgery involves an inflammatory response and can lead to the development of chronic pain. Neurotropin® (NTP) is a nonprotein extract of inflamed skin of rabbits inoculated with vaccinia virus. It has been widely used for the treatment of chronic pain. However, the in vivo effects of NTP on painful scar formation have not been determined. To investigate the molecular mechanisms underlying the effects of NTP on the inflammatory response, we evaluated gene expression in the scar tissues and dorsal root ganglions (DRGs) of mice administered NTP and control mice. Methods and results: Mice injected with saline or NTP were used as controls; other mice were subjected to surgery on the left hind paw to induce painful scar formation, and then injected with saline or NTP. Hind paw pain was evaluated by measuring the threshold for mechanical stimulation using the von Frey test. The paw withdrawal threshold gradually returned to pre-operative levels over 4 weeks post-operation; NTP-treated mice showed a significantly shortened recovery time of approximately 3 weeks, suggesting that NTP exerted an analgesic effect in this mouse model. Total RNA was extracted from the scarred hind paw tissues and DRGs were collected 1 week post-operation for a microarray analysis. Gene set enrichment analysis revealed that the expression of some gene sets related to inflammatory responses was activated or inhibited following surgery and NTP administration. Quantitative real-time reverse transcription-polymerase chain reaction analysis results for several genes were consistent with the microarray results. Conclusion: The administration of NTP to the hind paws of mice with painful scar formation following surgery diminished nociceptive pain and reduced the inflammatory response. NTP inhibited the expression of some genes involved in the response to surgery-induced inflammation. Therefore, NTP is a potential therapeutic option for painful scar associated with chronic pain.

Osteogenic Competence and Potency of the Bone Induction Principle: Inductive Substrates That Initiate "Bone: Formation by Autoinduction".

ABSTRACT: The de novo induction of bone has always been a fascinating phenomenon, keeping skeletal reconstructionists and cellular developmental biologists continuously engaged to finally provide a molecular and cellular approach to the induction of bone formation. A significant advancement was made by the purification and cloning of the human recombinant bone morphogenetic proteins, members of the transforming growth factor-ß supergene family. Human bone morphogenetic proteins are powerful inducers of bone in animal models including nonhuman primates. Translation in clinical contexts has however, proven to be surprisingly difficult. This review also describes the significant induction of bone formation by the human transforming growth factor-ß3 when implanted in heterotopic intramuscular sites of the Chacma baboon Papio ursinus. Large mandibular defects implanted with 250 mg human transforming growth factor-ß3 in human patients showed significant osteoinduction; however, the induction of bone was comparatively less than the induction of bone in P ursinus once again highlighting the conundrum of human osteoinduction: is the bone induction principle failing clinical translation?

Discovery of bone morphogenetic proteins - A historical perspective.

Bone morphogenetic proteins (BMPs) were purified from demineralized bone matrix by their ability to induce new bone formation in vivo. BMPs represent a large sub-family of proteins structurally related to TGF-beta and activins. Two BMP bone graft substitutes, BMP2 (InFuse®) and BMP7 (OP1®) have been developed as products for the repair of long bone non-union fractures and lumbar spinal fusion in humans. The approval of BMP2 and BMP7 based products for use in the clinic supports that the signals responsible for bone formation at ectopic sites can form a basis as therapeutics for bone repair and regeneration. This article describes a historical perspective of the discovery BMPs.

Pulsed Electromagnetic Fields and Tissue Engineering of the Joints.

BACKGROUND: Bone and joint formation, maintenance, and regeneration are regulated by both chemical and physical signals. Among the physical signals there is an increasing realization of the role of pulsed electromagnetic fields (PEMF) in the treatment of nonunions of bone fractures. The discovery of the piezoelectric properties of bone by Fukada and Yasuda in 1953 in Japan established the foundation of this field. Pioneering research by Bassett and Brighton and their teams resulted in the approval by the Food and Drug Administration (FDA) of the use of PEMF in the treatment of fracture healing. Although PEMF has potential applications in joint regeneration in osteoarthritis (OA), this evolving field is still in its infancy and offers novel opportunities. METHODS: We have systematically reviewed the literature on the influence of PEMF in joints, including articular cartilage, tendons, and ligaments, of publications from 2000 to 2016. CONCLUSIONS: PEMF stimulated chondrocyte proliferation, differentiation, and extracellular matrix synthesis by release of anabolic morphogens such as bone morphogenetic proteins and anti-inflammatory cytokines by adenosine receptors A2A and A3 in both in vitro and in vivo investigations. It is noteworthy that in clinical translational investigations a beneficial effect was observed on improving function in OA knees. However, additional systematic studies on the mechanisms of action of PEMF on joints and tissues therein, articular cartilage, tendons, and ligaments are required.

Optimization of Methods for Articular Cartilage Surface Tissue Engineering: Cell Density and Transforming Growth Factor Beta Are Critical for Self-Assembly and Lubricin Secretion.

OBJECTIVE: Lubricin/superficial zone protein (SZP)/proteoglycan4 (PRG4) plays an important role in boundary lubrication in articular cartilage. Lubricin is secreted by superficial zone chondrocytes and synoviocytes of the synovium. The specific objective of this investigation is to optimize the methods for tissue engineering of articular cartilage surface. The aim of this study is to investigate the effect of cell density on the self-assembly of superficial zone chondrocytes and lubricin secretion as a functional assessment. DESIGN: Superficial zone chondrocytes were cultivated as a monolayer at low, medium, and high densities. Chondrocytes at the three different densities were treated with transforming growth factor beta (TGF-ß)1 twice a week or daily, and the accumulated lubricin in the culture medium was analyzed by immunoblots and quantitated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Cell numbers in low and medium densities were increased by TGF-ß1; whereas cell numbers in high-density cell cultures were decreased by twice-a-week treatment of TGF-ß1. On the other hand, the cell numbers were maintained by daily TGF-ß treatment. Immunoblots and quantitation of lubricin by ELISA analysis indicated that TGF-ß1 stimulated lubricin secretion by superficial zone chondrocytes at all densities with twice-a-week TGF-ß treatment. It is noteworthy that the daily treatment of TGF-ß1 increased lubricin much higher compared with twice-a-week treatment. CONCLUSIONS: These data demonstrate that daily treatment is optimal for the TGF-ß1 response in a higher density of monolayer cultures. These findings have implications for self-assembly of surface zone chondrocytes of articular cartilage for application in tissue engineering of articular cartilage surface.

Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton.

Synovial joints are crucial for support and locomotion in vertebrates, and are the frequent site of serious skeletal defects and degenerative diseases in humans. Growth and differentiation factor 5 (Gdf5) is one of the earliest markers of joint formation, is required for normal joint development in both mice and humans, and has been genetically linked to risk of common osteoarthritis in Eurasian populations. Here, we systematically survey the mouse Gdf5 gene for regulatory elements controlling expression in synovial joints. We identify separate regions of the locus that control expression in axial tissues, in proximal versus distal joints in the limbs, and in remarkably specific sub-sets of composite joints like the elbow. Predicted transcription factor binding sites within Gdf5 regulatory enhancers are required for expression in particular joints. The multiple enhancers that control Gdf5 expression in different joints are distributed over a hundred kilobases of DNA, including regions both upstream and downstream of Gdf5 coding exons. Functional rescue tests in mice confirm that the large flanking regions are required to restore normal joint formation and patterning. Orthologs of these enhancers are located throughout the large genomic region previously associated with common osteoarthritis risk in humans. The large array of modular enhancers for Gdf5 provide a new foundation for studying the spatial specificity of joint patterning in vertebrates, as well as new candidates for regulatory regions that may also influence osteoarthritis risk in human populations.

Modulation of Superficial Zone Protein/Lubricin/PRG4 by Kartogenin and Transforming Growth Factor-ß1 in Surface Zone Chondrocytes in Bovine Articular Cartilage.

OBJECTIVE: Superficial zone protein (SZP)/lubricin/PRG4 functions as a boundary lubricant in articular cartilage to decrease friction and wear. As articular cartilage lubrication is critical for normal joint function, the accumulation of SZP at the surface of cartilage is important for joint homeostasis. Recently, a heterocyclic compound called kartogenin (KGN) was found to induce chondrogenic differentiation and enhance mRNA expression of lubricin. The objective of this study was to determine whether KGN can stimulate synthesis of SZP in superficial zone, articular chondrocytes. DESIGN: We investigated the effects of KGN and transforming growth factor-ß1 (TGF-ß1) on articular cartilage and synovium of the bovine knee joint by evaluating SZP secretion by enzyme-linked immunosorbent assay analysis. Monolayer, micromass, and explant cultures of articular cartilage, and monolayer culture of synoviocytes, were treated with KGN. SZP accumulation in the medium was evaluated and mRNA expression was measured through quantitative polymerase chain reaction. RESULTS: TGF-ß1 stimulated SZP secretion by superficial zone chondrocytes in monolayer, explant, and micromass cultures as expected. In addition, SZP secretion was inhibited by IL-1ß in explant cultures, and enhanced by TGF-ß1 in synoviocyte monolayer cultures. Although KGN elicited a 1.2-fold increase in SZP mRNA expression in combination with TGF-ß1, KGN neither stimulated any significant increases in SZP synthesis nor prevented catabolic decreases in SZP production from IL-1ß. CONCLUSIONS: These data suggest that the chondrogenic effects of KGN depend on cellular phenotype and differentiation status, as KGN did not alter SZP synthesis in differentiated, superficial zone articular chondrocytes.

Superficial Zone Extracellular Matrix Extracts Enhance Boundary Lubrication of Self-Assembled Articular Cartilage.

OBJECTIVE: Previous work has shown that increasing the production of boundary lubricant, superficial zone protein (SZP), did not reduce the friction coefficient of self-assembled articular cartilage constructs and was possibly due to poor retention of the lubricant. The aim of this investigation was to reduce the friction coefficient of self-assembled articular cartilage constructs through enhancing SZP retention by the exogenous addition of extracellular matrix (ECM) extracted from the superficial zone of native articular cartilage. DESIGN: Superficial zone cartilage was shaved from juvenile bovine femoral condyles using a dermatome, minced finely with razor blades, extracted with 4 M guanidine-hydrochloride, buffer exchanged with culture medium, and added directly to the culture medium of self-assembled articular cartilage constructs at low (10 µg/mL) and high (100 µg/mL) concentrations for 4 weeks. Biochemical and biomechanical properties were determined at the conclusion of 4 weeks culture. RESULTS: ECM treatment increased compressive and tensile stiffness of self-assembled articular cartilage constructs and decreased the friction coefficient. Glycosaminoglycan content decreased and collagen content increased significantly in self-assembled constructs by the ECM treatment. CONCLUSIONS: Friction coefficients of self-assembled articular cartilage constructs were reduced by adding extracted superficial zone ECM into the culture medium of self-assembled articular cartilage constructs.

Platelet-Rich Plasma Modulates Actions on Articular Cartilage Lubrication and Regeneration.

Osteoarthritis (OA) is characterized by articular cartilage degeneration in the joints and results in pain, swelling, stiffness, muscle atrophy, and attendant functional disability. The progression of OA cannot be prevented by drugs. Current treatments for OA focus on symptomatic relief of pain. Recently, platelet-rich plasma (PRP) injection has attracted much attention for modulating articular cartilage and synovial membrane in OA. Despite the increasing interest in PRP for the treatment of OA, the precise mechanisms underlying the actions of PRP on OA remain unclear. We have reviewed the known actions of PRP in the joint. The in vitro and in vivo evidence is reviewed concerning the potential of the modulation of PRP on normal articular cartilage and OA progression. PRP modulates the repair and regeneration of damaged articular cartilage in the joints and delays the degeneration of cartilage by stimulation of mesenchymal stem cell migration, proliferation, and differentiation into articular chondrocytes. In addition to the symptomatic relief, PRP is a biological response modifier of inflammatory nuclear factor-κB signaling pathway and PRP reduces the pain by decreasing inflammation and angiogenesis of the synovial membrane where pain receptors are localized. PRP has the therapeutic potential not only to promote tissue regeneration, but also to contribute to articular cartilage lubrication by decreasing the friction coefficient and minimizing wear. Although further refinements and improvements are needed in standardized PRP preparations, PRP may modulate regeneration of articular cartilage and retards the progression of OA by stimulating cell migration, proliferation, differentiation of progenitor/stem cells, joint homeostasis, and joint lubrication.

BMP-7 and Bone Regeneration: Evaluation of Dose-Response in a Rodent Segmental Defect Model.

OBJECTIVES: To develop and validate a translatable and reproducible rodent critical-sized defect (CSD) model and to determine the optimal dose of recombinant human bone morphogenetic protein (BMP)-7 required to consistently heal the CSD in the new model. METHODS: Rats with 6-mm CSDs stabilized with a commercial radiolucent plate and screws with angular stability were randomly assigned to 4 treatment groups with varied doses of recombinant human BMP-7 (25, 50, 75, and 100 µg) on absorbable collagen sponge and a single control group (absorbable collagen sponge alone). Bone formation was evaluated by radiographs, micro-computed tomography, histology, and biomechanics. RESULTS: All the rats treated with 100 µg of BMP-7 with CSDs were united by 4 weeks and all 75- and 50-µg-group rats united by 6 weeks. None of the animals in the 25-µg BMP-7 group or the control group were healed at the time of killing. Bone volume, bone mineral density, the ratio of bone volume to total volume, stiffness, and ultimate load to failure were maximal in the 50-µg group. Total callus volume progressively increased with increasing BMP dose. Histologic analysis demonstrated increased callus width with increasing BMP-7 doses above 50 µg, but the bone seemed structurally abnormal. CONCLUSIONS: There was a 100% union rate in the 50-, 75-, and 100-µg BMP-7-treated groups. None of the control or 25-µg-dose rats united. The biomechanical data demonstrated that 50 µg of BMP-7 produced the highest mechanical strength in the bone regenerate. These data also suggest that administration of BMP-7 above 50 µg does not improve bone regeneration and actually seems to produce lower quality bone with diminished biomechanical properties.

The distribution of superficial zone protein (SZP)/lubricin/PRG4 and boundary mode frictional properties of the bovine diarthrodial joint.

The diarthrodial, knee joint is a remarkably efficient bearing system; articulating cartilage surfaces provide nearly frictionless performance with minimal wear. The low friction properties of the cartilage surfaces are due in part to the boundary lubricant, superficial zone protein (SZP); also known as lubricin or proteoglycan 4 (PRG4). In previous work, SZP localization and cartilage friction were examined across the femoral condyles. Studies in the literature have also individually investigated the other tissues that comprise the human knee and four-legged animal stifle joint, such as the meniscus or patella. However, comparisons between individual studies are limited due to the variable testing conditions employed. Friction is a system property that is dependent on the opposing articulating surface, entraining speed, and loading. A cross-comparison of the frictional properties and SZP localization across the knee/stifle joint tissues utilizing a common testing configuration is therefore needed. The objective of this investigation was to determine the friction coefficient and SZP localization of the tissues comprising the three compartments of the bovine stifle joint: patella, patellofemoral groove, femoral condyles, meniscus, tibial plateau, and anterior cruciate ligament. The boundary mode coefficient of friction was greater in tissues of the patellofemoral compartment than the lateral and medial tibiofemoral compartments. SZP immunolocalization followed this trend with reduced depth of staining and intensity in the patella and patellofemoral groove compared to the femoral condyles and tibial plateau. These results illustrate the important role of SZP in reducing friction in the tissues and compartments of the knee/stifle joint.

Regeneration of Articular Cartilage Surface: Morphogens, Cells, and Extracellular Matrix Scaffolds.

The articular cartilage is a well-organized tissue for smooth and friction-free joint movement for locomotion in animals and humans. Adult articular cartilage has a very low self-regeneration capacity due to its avascular nature. The regeneration of articular cartilage surface is critical to prevent the progression to osteoarthritis (OA). Although various joint resurfacing procedures in experimental articular cartilage defects have been developed, no standardized clinical protocol has yet been established. The three critical ingredients for tissue regeneration are morphogens and growth factors, cells, and scaffolds. The concepts based on the regeneration triad have been extensively investigated in animal models. However, these studies in animal models have demonstrated variable results and outcomes. An optimal animal model must precisely mimic and model the sequence of events in articular cartilage regeneration in human. In this article, the progress and remaining challenges in articular cartilage regeneration in animal models are reviewed. The role of individual morphogens and growth factors in cartilage regeneration has been investigated. In normal articular cartilage homeostasis, morphogens and growth factors function sequentially in tissue regeneration. Mesenchymal stem cell-based repair of articular cartilage defects, performed with or without various growth factors and scaffolds, has been widely attempted in animal models. Stem cells, including embryonic and adult stem cells and induced pluripotent stem cells, have also been reported as attractive cell sources for articular cartilage surface regeneration. Several studies with regard to scaffolds have been advanced, including recent investigations based on nanomaterials, functional mechanocompatible scaffolds, multilayered scaffolds, and extracellular matrix scaffolds for articular cartilage surface regeneration. Continuous refinement of animal models in chondral and osteochondral defects provide opportunities that support further advances in tissue engineering for the optimal articular cartilage surface regeneration.

Thyroid hormones enhance the biomechanical functionality of scaffold-free neocartilage.

INTRODUCTION: The aim of this study was to investigate the effects of thyroid hormones tri-iodothyronine (T3), thyroxine (T4), and parathyroid hormone (PTH) from the parathyroid glands, known to regulate the developing limb and growth plate, on articular cartilage tissue regeneration using a scaffold-free in vitro model. METHODS: In Phase 1, T3, T4, or PTH was applied during weeks 1 or 3 of a 4-week neocartilage culture. Phase 2 employed T3 during week 1, followed by PTH during week 2, 3, or weeks 2 to 4, to further enhance tissue properties. Resultant neotissues were evaluated biochemically, mechanically, and histologically. RESULTS: In Phase 1, T3 and T4 treatment during week 1 resulted in significantly enhanced collagen production; 1.4- and 1.3-times untreated neocartilage. Compressive and tensile properties were also significantly increased, as compared to untreated and PTH groups. PTH treatment did not result in notable tissue changes. As T3 induces hypertrophy, in Phase 2, PTH (known to suppress hypertrophy) was applied sequentially after T3. Excitingly, sequential treatment with T3 and PTH reduced expression of hypertrophic marker collagen X, while yielding neocartilage with significantly enhanced functional properties. Specifically, in comparison to no hormone application, these hormones increased compressive and tensile moduli 4.0-fold and 3.1-fold, respectively. CONCLUSIONS: This study demonstrated that T3, together with PTH, when applied in a scaffold-free model of cartilage formation, significantly enhanced functional properties. The novel use of these thyroid hormones generates mechanically robust neocartilage via the use of a scaffold-free tissue engineering model.

Stimulation of the superficial zone protein and lubrication in the articular cartilage by human platelet-rich plasma.

BACKGROUND: Platelet-rich plasma (PRP) contains high concentrations of autologous growth factors that originate from platelets. Intra-articular injections of PRP have the potential to ameliorate the symptoms of osteoarthritis in the knee. Superficial zone protein (SZP) is a boundary lubricant in articular cartilage and plays an important role in reducing friction and wear and therefore is critical in cartilage homeostasis. PURPOSE: To determine if PRP influences the production of SZP from human joint-derived cells and to evaluate the lubricating properties of PRP on normal bovine articular cartilage. STUDY DESIGN: Controlled laboratory study. METHODS: Cells were isolated from articular cartilage, synovium, and the anterior cruciate ligament (ACL) from 12 patients undergoing ACL reconstruction. The concentrations of SZP in PRP and culture media were measured by enzyme-linked immunosorbent assay. Cellular proliferation was quantified by determination of cell numbers. The lubrication properties of PRP from healthy volunteers on bovine articular cartilage were investigated using a pin-on-disk tribometer. RESULTS: In general, PRP stimulated proliferation in cells derived from articular cartilage, synovium, and ACL. It also significantly enhanced SZP secretion from synovium- and cartilage-derived cells. An unexpected finding was the presence of SZP in PRP (2.89 ± 1.23 µg/mL before activation and 3.02 ± 1.32 µg/mL after activation). In addition, under boundary mode conditions consisting of high loads and low sliding speeds, nonactivated and thrombin-activated PRP decreased the friction coefficient (µ = 0.012 and µ = 0.015, respectively) compared with saline (µ = 0.047, P < .004) and high molecular weight hyaluronan (µ = 0.080, P < .006). The friction coefficient of the cartilage with PRP was on par with that of synovial fluid. CONCLUSION: PRP significantly stimulates cell proliferation and SZP secretion by articular cartilage and synovium of the human knee joint. Furthermore, PRP contains endogenous SZP and, in a functional bioassay, lubricates bovine articular cartilage explants. CLINICAL RELEVANCE: These findings provide evidence to explain the biochemical and biomechanical mechanisms underlying the efficacy of PRP treatment for osteoarthritis or damage in the knee joint.

Stimulation of Superficial Zone Protein/Lubricin/PRG4 by Transforming Growth Factor-ß in Superficial Zone Articular Chondrocytes and Modulation by Glycosaminoglycans.

Superficial zone protein (SZP), also known as lubricin and proteoglycan 4 (PRG4), plays an important role in the boundary lubrication of articular cartilage and is regulated by transforming growth factor (TGF)-ß. Here, we evaluate the role of cell surface glycosaminoglycans (GAGs) during TGF-ß1 stimulation of SZP/lubricin/PRG4 in superficial zone articular chondrocytes. We utilized primary monolayer superficial zone articular chondrocyte cultures and treated them with various concentrations of TGF-ß1, in the presence or absence of heparan sulfate (HS), heparin, and chondroitin sulfate (CS). The cell surface GAGs were removed by pretreatment with either heparinase I or chondroitinase-ABC before TGF-ß1 stimulation. Accumulation of SZP/lubricin/PRG4 in the culture medium in response to stimulation with TGF-ß1 and various exogenous GAGs was demonstrated by immunoblotting and quantitated by enzyme-linked immunosorbent assay. We show that TGF-ß1 and exogenous HS enhanced SZP accumulation of superficial zone chondrocytes in the presence of surface GAGs. At the dose of 1 ng/mL of TGF-ß1, the presence of exogenous heparin inhibited SZP accumulation whereas the presence of exogenous CS stimulated SZP accumulation in the culture medium. Enzymatic depletion of GAGs on the surface of superficial zone chondrocytes enhanced the ability of TGF-ß1 to stimulate SZP accumulation in the presence of both exogenous heparin and CS. Collectively, these results suggest that GAGs at the surface of superficial zone articular chondrocytes influence the response to TGF-ß1 and exogenous GAGs to stimulate SZP accumulation. Cell surface GAGs modulate superficial zone chondrocytes' response to TGF-ß1 and exogenous HS.

Surface zone articular chondrocytes modulate the bulk and surface mechanical properties of the tissue-engineered cartilage.

The central hypothesis of functional tissue engineering is that an engineered construct can serve as a viable replacement tissue in vivo by replicating the structure and function of native tissue. In the case of articular cartilage, this requires the reproduction of the bulk mechanical and surface lubrication properties of native hyaline cartilage. Cartilage tissue engineering has primarily focused on achieving the bulk mechanical properties of native cartilage such as the compressive aggregate modulus and tensile strength. A scaffold-free self-assembling process has been developed that produces engineered cartilage with compressive properties approaching native tissue levels. Thus, the next step in this process is to begin addressing the friction coefficient and wear properties of these engineered constructs. The superficial zone protein (SZP), also known as lubricin or PRG4, is a boundary mode lubricant that is synthesized by surface zone (SZ) articular chondrocytes. Under conditions of high loading and low sliding speeds, SZP reduces friction and wear at the articular surface. The objective of this investigation was to determine whether increasing the proportion of SZ chondrocytes in cartilage constructs, in the absence of external stimuli such as growth factors and mechanical loading, would enhance the secretion of SZP and improve their frictional properties. In this study, cartilage constructs were engineered through a self-assembling process with varying ratios of SZ and middle zone (MZ) chondrocytes (SZ:MZ): 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs containing different ratios of SZ and MZ chondrocytes did not significantly differ in the glycosaminoglycan composition or compressive aggregate modulus. In contrast, tensile properties and collagen content were enhanced in nearly all constructs containing greater amounts of SZ chondrocytes. Increasing the proportion of SZ chondrocytes had the hypothesized effect of improving the synthesis and secretion of SZP. However, increasing the SZ chondrocyte fraction did not significantly reduce the friction coefficient. These results demonstrate that additional factors, such as SZP-binding macromolecules, surface roughness, and adhesion, need to be examined to modulate the lubrication properties of engineered cartilage.

Immunohistochemical Localization of Bone Morphogenetic Proteins (BMPs) and their Receptors in Solitary and Multiple Human Osteochondromas.

The expression of bone morphogenetic proteins (BMPs) and their cognate receptors (BMPRs) in osteochondromas has not been investigated. We determined the immunohistochemical localization and distribution of BMP-2/4, -6 and -7; BMP receptors BMPR-1A, BMPR-1B and BMPR-2; signal transducing proteins phosphorylated Smad1/5/8; and BMP antagonist noggin in the cartilaginous cap of solitary (SO) and multiple (MO) human osteochondromas and compared these with bovine growth plate and articular cartilage. The distribution and localization patterns for BMP-6, BMP-7, BMPR-1A and BMPR-2 were similar between the cartilaginous cap and the growth plate. BMP-2/4 and BMPR-1B were present throughout the growth plate. However, BMP-2/4 and phosphorylated Smad1/5/8 were mainly detected in proliferating chondrocytes of the cartilaginous cap. Also, BMPR-1B was found in hypertrophic chondrocytes of SO and proliferating chondrocytes of MO. Noggin was observed in resting chondrocytes and, to a lesser extent, in clustered proliferating chondrocytes in SO. On the other hand, noggin in MO was observed in proliferating chondrocytes. Since BMPs can stimulate proliferation and hypertrophic differentiation of chondrocytes, these findings suggest that there is an imbalance of BMP-2/4 and noggin interactions that may lead to abnormal regulation of chondrocyte proliferation and differentiation in the cartilaginous cap of human osteochondromas.

Transforming growth factor ß-induced superficial zone protein accumulation in the surface zone of articular cartilage is dependent on the cytoskeleton.

The phenotype of articular chondrocytes is dependent on the cytoskeleton, specifically the actin microfilament architecture. Articular chondrocytes in monolayer culture undergo dedifferentiation and assume a fibroblastic phenotype. This process can be reversed by altering the actin cytoskeleton by treatment with cytochalasin. Whereas dedifferentiation has been studied on chondrocytes isolated from the whole cartilage, the effects of cytoskeletal alteration on specific zones of cells such as superficial zone chondrocytes are not known. Chondrocytes from the superficial zone secrete superficial zone protein (SZP), a lubricating proteoglycan that reduces the coefficient of friction of articular cartilage. A better understanding of this phenomenon may be useful in elucidating chondrocyte dedifferentiation in monolayer and accumulation of the cartilage lubricant SZP, with an eye toward tissue engineering functional articular cartilage. In this investigation, the effects of cytoskeletal modulation on the ability of superficial zone chondrocytes to secrete SZP were examined. Primary superficial zone chondrocytes were cultured in monolayer and treated with a combination of cytoskeleton modifying reagents and transforming growth factor ß (TGFß) 1, a critical regulator of SZP production. Whereas cytochalasin D maintains the articular chondrocyte phenotype, the hallmark of the superficial zone chondrocyte, SZP, was inhibited in the presence of TGFß1. A decrease in TGFß1-induced SZP accumulation was also observed when the microtubule cytoskeleton was modified using paclitaxel. These effects of actin and microtubule alteration were confirmed through the application of jasplakinolide and colchicine, respectively. As Rho GTPases regulate actin organization and microtubule polymerization, we hypothesized that the cytoskeleton is critical for TGFß-induced SZP accumulation. TGFß-mediated SZP accumulation was inhibited by small molecule inhibitors ML141 (Cdc42), NSC23766 (Rac1), and Y27632 (Rho effector Rho Kinase). On the other hand, lysophosphatidic acid, an upstream activator of Rho, increased SZP synthesis in response to TGFß1. These results suggest that SZP production is dependent on the functional cytoskeleton, and Rho GTPases contribute to SZP accumulation by modulating the actions of TGFß.

Local control of hepatic phenotype with growth factor-encoded surfaces.

The goal of the present study was to modulate the phenotype expression of hepatocytes in vitro on surfaces imprinted with growth factors (GFs). Hepatocyte growth factor (HGF) or transforming-growth factor-ß1 (TGF-ß1) were mixed with collagen (I) and robotically printed onto standard glass slides to create arrays of 300 µm or 500 µm diameter spots. Primary rat hepatocytes were seeded on top of the arrays, forming clusters corresponding in size to the underlying protein spots. The TGF-ß1 spots appeared to downregulate markers of hepatic (epithelial) phenotype while upregulating expression of mesenchymal markers. Conversely, hepatocytes cultured on HGF spots maintained high level of epithelial markers. When hepatocytes were seeded onto alternating spots of HGF and TGF-ß1, their phenotype was found to depend on center-to-center distance between the spots. At shorter distances cross-expression of epithelial and mesenchymal markers was observed while at distances exceeding 1.25 mm divergence of phenotypes, epithelial on HGF and mesenchymal on TGF-ß was seen. Overall, our results demonstrate that GF-encoded surfaces can modulate phenotype within groups of cells cultured on the same surface. Given the importance of phenotype switching in development, fibrosis and cancer, this platform may be used to gain useful insights into the mechanisms of processes such as epithelial-to-mesenchymal transition or stem cell fate selections.

Distinct patterns of gene expression in the superficial, middle and deep zones of bovine articular cartilage.

Hyaline articular cartilage will not heal spontaneously, and lesions in hyaline articular cartilage often result in degenerative joint disease. Considerable progress has been made with respect to the responsive stem cells, inductive signals and extracellular scaffolding required for the optimal regeneration of cartilage. However, many challenges remain, such as topographic differences in the functional zones of articular cartilage. We hypothesized that a distinct set of differentially expressed genes define the surface, middle and deep zones of hyaline articular cartilage. Microarray analysis of bovine articular cartilage from the superficial and middle zones revealed 52 genes differentially expressed ≥ 10-fold and 114 additional genes differentially expressed ≥ five-fold. However, no genes were identified with a ≥ five-fold difference in expression when comparing articular cartilage from the middle and deep zones. There are distinct, differential gene expression patterns in the superficial and middle zones of hyaline articular cartilage that highlight the functional differences between these zones. This investigation has implications for the tissue engineering and regeneration of hyaline articular cartilage.