Periosteal Chondrogenesis Research Articles

Does periosteum promote chondrogenesis? A comparison of free periosteal and perichondrial grafts in the regeneration of ear cartilage.

BackgroundElastic ear cartilage is a good source of tissue for support or augmentation in plastic and reconstructive surgery. However, the amount of ear cartilage is limited and excessive use of cartilage can cause deformation of the auricular framework. This animal study investigated the potential of periosteal chondrogenesis in an ear cartilage defect model.MethodsTwelve New Zealand white rabbits were used in the present study. Four ear cartilage defects were created in both ears of each rabbit, between the central artery and marginal veins. The defects were covered with perichondrium (group 1), periosteum taken from the calvarium (group 2), or periosteum taken from the tibia (group 3). No coverage was performed in a control group (group 4). All animals were sacrificed 6 weeks later, and the ratio of neo-cartilage to defect size was measured.ResultsSignificant chondrogenesis occurred only in group 1 (cartilage regeneration ratio: mean± standard deviation, 0.97± 0.60), whereas the cartilage regeneration ratio was substantially lower in group 2 (0.10± 0.11), group 3 (0.08± 0.09), and group 4 (0.08± 0.14) (p=0.004). Instead of chondrogenesis, osteogenesis was observed in the periosteal graft groups. No statistically significant differences were found in the amount of osteogenesis or chondrogenesis between groups 2 and 3. Group 4 showed fibrous tissue accumulation in the defect area.ConclusionPeriosteal grafts showed weak chondrogenic potential in an ear cartilage defect model of rabbits; instead, they exhibited osteogenesis, irrespective of their embryological origin.

Aggrecan and COMP Improve Periosteal Chondrogenesis by Delaying Chondrocyte Hypertrophic Maturation.

The generation of cartilage from progenitor cells for the purpose of cartilage repair is often hampered by hypertrophic differentiation of the engineered cartilaginous tissue caused by endochondral ossification. Since a healthy cartilage matrix contains high amounts of Aggrecan and COMP, we hypothesized that their supplementation in the biogel used in the generation of subperiosteal cartilage mimics the composition of the cartilage extracellular matrix environment, with beneficial properties for the engineered cartilage. Supplementation of COMP or Aggrecan was studied in vitro during chondrogenic differentiation of rabbit periosteum cells and periosteum-derived chondrocytes. Low melting agarose was supplemented with bovine Aggrecan, human recombinant COMP or vehicle and was injected between the bone and periosteum at the upper medial side of the tibia of New Zealand white rabbits. Generated subperiosteal cartilage tissue was analyzed for weight, GAG and DNA content and ALP activity. Key markers of different phases of endochondral ossification were measured by RT-qPCR. For the in vitro experiments, no significant differences in chondrogenic marker expression were detected following COMP or Aggrecan supplementation, while in vivo favorable chondrogenic marker expression was detected. Gene expression levels of hypertrophic markers as well as ALP activity were significantly decreased in the Aggrecan and COMP supplemented conditions compared to controls. The wet weight and GAG content of the in vivo generated subperiosteal cartilage tissue was not significantly different between groups. Data demonstrate the potential of Aggrecan and COMP to favorably influence the subperiosteal microenvironment for the in vivo generation of cartilage for the optimization of cartilage regenerative approaches.

Temporary inhibition of the plasminogen activator inhibits periosteal chondrogenesis and promotes periosteal osteogenesis during appendicular bone fracture healing

Aminocaproic acid is approved as an anti-fibrinolytic for use in joint replacement and spinal fusion surgeries to limit perioperative blood loss. Previous animal studies have demonstrated a pro-osteogenic effect of aminocaproic acid in spine fusion models. Here, we tested if aminocaproic acid enhances appendicular bone healing and we sought to uncover the effect of aminocaproic acid on osteoprogenitor cells (OPCs) during bone regeneration. We employed a well-established murine femur fracture model in adult C57BL/6J mice after receiving two peri-operative injections of aminocaproic acid. Routine histological assays, biomechanical testing and micro-CT analyses were utilized to assess callus volume, and strength, progenitor cell proliferation, differentiation, and remodeling in vivo. Two disparate ectopic transplantation models were used to study the effect of the growth factor milieu within the early fracture hematoma on osteoprogenitor cell fate decisions. Aminocaproic acid treated femur fractures healed with a significantly smaller cartilaginous callus, and this effect was also observed in the ectopic transplantation assays. We hypothesized that aminocaproic acid treatment resulted in a stabilization of the early fracture hematoma, leading to a change in the growth factor milieu created by the early hematoma. Gene and protein expression analysis confirmed that aminocaproic acid treatment resulted in an increase in Wnt and BMP signaling and a decrease in TGF-β-signaling, resulting in a shift from chondrogenic to osteogenic differentiation in this model of endochondral bone formation. These experiments demonstrate for the first time that inhibition of the plasminogen activator during fracture healing using aminocaproic acid leads to a change in cell fate decision of periosteal osteoprogenitor cells, with a predominance of osteogenic differentiation, resulting in a larger and stronger bony callus. These findings may offer a promising new use of aminocaproic acid, which is already FDA-approved and offers a very safe risk profile.

The periosteal requirement and temporal dynamics of BMP2‐induced middle phalanx regeneration in the adult mouse

Regeneration of mammalian limbs is restricted to amputation of the distal digit tip, the terminal phalanx (P3). The adjacent skeletal element, the middle phalanx (P2), has emerged as a model system to investigate regenerative failure and as a site to test approaches aimed at enhancing regeneration. We report that exogenous application of bone morphogenetic protein 2 (BMP2) stimulates the formation of a transient cartilaginous callus distal to the amputation plane that mediates the regeneration of the amputated P2 bone. BMP2 initiates a significant regeneration response during the periosteal‐derived cartilaginous healing phase of P2 bone repair, yet fails to induce regeneration in the absence of periosteal tissue, or after boney callus formation. We provide evidence that a temporal component exists in the induced regeneration of P2 that we define as the “regeneration window.” In this window, cells are transiently responsive to BMP2 after the amputation injury. Simple re‐injury of the healed P2 stump acts to reinitiate endogenous bone repair, complete with periosteal chondrogenesis, thus reopening the “regeneration window” and thereby recreating a regeneration‐permissive environment that is responsive to exogenous BMP2 treatment.

A novel recirculating flow-perfusion bioreactor for periosteal chondrogenesis

Previous research indicated that engineered cartilage was soft and fragile due to less extracellular matrix than native articular cartilage. Consequently, the focus of this study was mostly confined to application in vitro function. In order to generate 3D engineered cartilage resembling native articular cartilage, we developed a recirculating flow-perfusion bioreactor to simulate the motion of a native diarthrodial joint by offering shear stress and hydrodynamic pressure simultaneously. The bioreactor we developed offers steady oscillating laminar flow (maximum shear stress of 250 dyne/cm(2)) and hydrodynamic pressure (increased from 0 to 15 psi) simultaneously. The periosteal explants were harvested from the proximal medial tibiae of rabbits and fixed onto PCL scaffold with four corner sutures and cambium layer facing upward, then these periosteal composites (periosteum/ PCL) were placed into the culture chamber of our bioreactor for six weeks in vitro culture. The cartilage yield in our recirculating bioreactor was 75-85%. The outcome was better than the 65-75% in the spinner flask bioreactor (shear stress only) and 17% in static culture. In addition, there was a significant difference in the cell morphology and zonal organisation among the three methods of culture; the engineered cartilage in the recirculating bioreactor presented many more characteristics of native articular cartilage. If the environment of culture provides the shear stress and hydrodynamic pressure simultaneously, the composition of the engineered cartilage resembles native articular cartilage, including their ECM composition, cell distribution, zonal organisation and mechanical properties.

Directional fluid flow enhancesin vitroperiosteal tissue growth and chondrogenesis on poly‐ε‐caprolactone scaffolds

The purpose of this study was to investigate the effect of directional fluid flow on periosteal chondrogenesis. Periosteal explants were harvested from 2-month-old rabbits and sutured onto poly-epsilon-caprolactone (PCL) scaffolds with the cambium layer facing away from the scaffolds. The periosteum/PCL composites were cultured in suspension in spinner flask bioreactors and exposed to various fluid flow velocities: 0, 20, 60, and 150 rpm for 4 h each day for 6 weeks. The application of fluid flow significantly increased percent cartilage yield in periosteal explants from 17% in the static controls to 65-75% under fluid flow (there was no significant difference between 20, 60, or 150 rpm). The size of the neocartilage was also significantly greater in explants exposed to fluid flow compared with static culture. The development of zonal organization within the engineered cartilage was observed predominantly in the tissue exposed to flow conditions. The Young's modulus of the engineered cartilage exposed to 60 rpm was significantly greater than the samples exposed to 150 and 20 rpm. These results demonstrate that application of directional fluid flow to periosteal explants secured onto PCL scaffolds enhances cell proliferation, chondrogenic differentiation, and cell organization and alters the biomechanical properties of the engineered cartilage.

Transforming growth factor-β1 modulates insulin-like growth factor binding protein-4 expression and proteolysis in cultured periosteal explants

Objective Periosteum is involved in bone growth and fracture healing and has been used as a cell source and tissue graft for tissue engineering and orthopedic reconstruction including joint resurfacing. Periosteum can be induced by transforming growth factor beta (TGF-β) or insulin-like growth factor-I (IGF-I) alone or in combination to form cartilage. However, little is known about the interaction between IGF and TGF-β signaling during periosteal chondrogenesis. The purpose of this study was to determine the effect of TGF-β1 on IGF binding protein-4 (IGFBP-4) and the IGFBP-4 protease pregnancy-associated plasma protein-A (PAPP-A) expression in cultured periosteal explants. Design Periosteal explants from rabbits were cultured with or without TGF-β1. IGFBP-4 and PAPP-A mRNA levels were determined by real-time quantitative PCR. Conditioned medium was analyzed for IGFBP-4 and PAPP-A protein levels and IGFBP-4 protease activity. Results TGF-β1-treated explants contained lower IGFBP-4 mRNA levels throughout the culture period with a maximum reduction of 70% on day 5 of culture. Lower levels of IGFBP-4 protein were also detected in the conditioned medium from TGF-β1-treated explants. PAPP-A mRNA levels were increased 1.6-fold, PAPP-A protein levels were increased threefold, and IGFBP-4 protease activity was increased 8.5-fold between 7 and 10 days of culture (the onset of cartilage formation in this model) in conditioned medium from TGF-β1-treated explants. Conclusions This study demonstrates that TGF-β1 modulates the expression of IGFBP-4 and PAPP-A in cultured periosteal explants.

A novel in vivo model to study endochondral bone formation; HIF-1α activation and BMP expression

Numerous growth and transcription factors have been implicated in endochondral bone formation of the growth plate. Many of these factors are up-regulated during hypoxia and downstream of Hypoxia-Inducible Factor (HIF)-1α activation. However, the specific function of these factors, in the context of oxygenation and metabolic adaptation during adult periosteal endochondral bone formation, is largely unknown. Here, we studied HIF-1α and the possible roles of (HIF-1α related) growth and transcription factors in a recently developed in vivo model for adult periosteal endochondral bone formation. At different phases of periosteal endochondral bone formation, mRNA levels of Transforming Growth Factor (TGF)-β 1, Bone Morphogenetic Proteins (BMP)-2, -4, and -7, Indian Hedgehog (Ihh), Parathyroid Hormone-related Protein (PTHrP), Sox9, Runx2, HIF-1α, Vascular Endothelial Growth Factor (VEGF), periostin (POSTN), and Glyceraldehyde-3-Phophate Dehydrogenase (GAPDH) were evaluated with RT-real time-PCR. Also protein levels of TGF-β 1, BMP-2, -4, and -7, HIF-1α, and POSTN were examined. During the chondrogenic phase, the expression of Sox9, Ihh, and HIF-1α was significantly up-regulated. TGF-β 1 mRNA levels were rather constant, and the mRNA levels of BMPs were significantly lower. Immunohistochemical detection of corresponding gene products, however, revealed the presence of the proteins of TGF-β 1, BMP-2, -4, and -7, HIF-1α, and POSTN within the chondrocytes during chondrogenesis. This discrepancy in gene expression between mRNA and protein level for TGF-β1 and the different BMPs is indicative of post-transcriptional regulation of protein synthesis. HIF-1α activation and up-regulation of GAPDH support a hypoxia-induced metabolic shift during periosteal chondrogenesis. Our model recapitulates essential steps in osteochondrogenesis and provides a new experimental system to study and ultimately control tissue regeneration in the adult organism.

Clonal growth of human articular cartilage and the functional role of the periosteum in chondrogenesis

Clinical cartilage repair with transplantation of cultured chondrocytes, the first described technique introduced in 1994, includes a periosteal membrane but today cells are also implanted without the periosteal combination. The aim of this study was to see if the periosteum had more than a biomechanical function and if the periosteum had a biological effect on the seeded cells tested in an agarose system in which the clonal growth in agarose and the external growth stimulation could be analysed. Four different experiments were used to study the growth of human chondrocytes in agarose and the periosteal influence. Human chondrocytes were isolated and transferred to either primary or secondary agarose culture. After 4 weeks, the total number of clones >50 microm was counted. Cocultures of chondrocytes and periosteal tissue, cultures of chondrocytes with conditioned medium from chondrocytes, periosteal cells and fibroblast were used to study a potential stimulatory effect on growth and different cytokines and growth factors were analysed. It was found that the human chondrocytes had different growth properties in agarose with the formation of four different types of clones: a homogenous clone without matrix production, a homogenous clone with matrix production, a differentiated clone with matrix production and finally a differentiated clone without matrix production. The periosteum exerted a paracrine effect on cultured chondrocytes in agarose resulting in a higher degree of cloning. The chondrocytes produced significant amounts of interleukin (IL)-6, IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF) and transforming growth factor (TGF)-beta. The periosteum produced significant amounts of IL-6, IL-8 and TGF-beta. Cocultures of chondrocytes and periosteum demonstrated a potentiation of IL-6 and IL-8 release but not of TGF-beta and GM-CSF. Articular chondrocytes are able to form clones of different properties in agarose and the periosteum has a capacity of stimulating chondrocyte clonal growth and differentiation and secretes significant amounts of IL-6, IL-8, GM-CSF and TGF-beta. It may be that the repair of cartilage defects with seeded chondrocytes could benefit from the combination with a periosteal graft. The production of TGF-beta by implanted chondrocytes could influence the chondrogenic cells in the periosteum to start a periosteal chondrogenesis and together with the matrix from implanted chondrocyte production, a repair of cartilaginous appearance may develop; a dual chondrogenic response is possible.

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis.

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.

FGF-2 enhances TGF-β1-induced periosteal chondrogenesis

The use of periosteum as a cell source for the in vitro engineering of grafts for articular cartilage repair requires the development of methods to obtain high viable cell numbers in the early stages of culture. In this study, we demonstrate that the addition of a mitogen, fibroblast growth factor-2 (FGF-2), during the early stage of the in vitro culture of periosteum in the presence of transforming growth factor-β1 (TGF-β1), significantly enhances cell proliferation, which results in increased neo-cartilage formation at later stages. Periosteal explants were cultured in vitro within alginate or agarose based gels in the presence of either FGF-2 for the first week, TGF-β1 for the first 2 weeks, FGF-2 and TGF-β1 for the first week and first 2 weeks respectively, or no added factors. Consistent with previous studies, periosteum derived neo-chondrogenesis occurred only in the presence of TGF-β1. The neo-cartilage was found to contain cartilage specific proteoglycans and Type-II collagen as determined by safranin-O and immunohistochemical staining respectively. Further medium supplementation with FGF-2 stimulated early cell proliferation (>3 fold higher total DNA content per explant at day 10). This resulted in a marked increase in the size of the cultured explants and in the total area of the explant staining positive for safranin-O (from around 50% to 85%, ( p<0.05)) after 6 weeks culture. The ability to generate significant quantities of neo-cartilage within a biocompatible and biodegradable matrix such as alginate, which lacks the immunogenicity of agarose, could open new pathways to utilizing such constructs in articular cartilage tissue engineering applications.

Serum-free media for periosteal chondrogenesis in vitro

Organ culture studies involving whole explants of periosteum have been useful for studying chondrogenesis, but to date the standard culture model for these explants has required the addition of fetal bovine serum to the media. Numerous investigators have succeeded in culturing chondrocytes and embryonic cells in serum-free conditions but there have been no studies focused on achieving a defined, serum-free media for culturing periosteal explants. The purpose of the present investigation was to determine if whole periosteal explants can be grown and produce cartilage in serum-free conditions, and to define the minimum media supplements that would be conducive to chondrogenesis. 321 periosteal explants were obtained from the medial proximal tibiae of 31 two month-old NZ white rabbits and cultured using a published agarose suspension organ culture model and DMEM for six weeks. The explants were cultured with and without fetal bovine serum or bovine serum albumin and exposed to transforming growth factor beta alone, a combination of growth factors we call ChondroMix (10 ng/ml transforming growth factor beta, 50 ng/ml basic fibroblast growth factor, and 5 μg/ml growth hormone), and/or ITS+ (2.08 μg/ml each of insulin, transferrin, and selenious acid, plus 1.78 μg/ml linoleic acid and 0.42 mg/ml BSA). Maximal chondrogenic stimulation in this study was observed with the combination of ChondroMix and ITS+. However, the minimal requirement to match or exceed the level of chondrogenic stimulation seen in the standard model (TGF-1 in 10% FBS) was achieved simply by the addition of 2.0 μg/ml insulin in 0.1% BSA-containing medium ( p<0.05). Therefore, based on our results, it would be reasonable to assume that insulin is the component in ITS+ responsible for the observed increase in total cartilage growth. Lower concentrations of insulin were not effective, suggesting that the observed effect of insulin requires activation of the IGF-1 receptor.

Potential healing benefit of an osteoperiosteal bone plug from the proximal tibia on a mosaicplasty donor-site defect in the knee. An experimental study in the goat.

Autologous osteochondral transplantation is a popular treatment for articular cartilage lesions in the knee joint. The donor defect is commonly left empty and remains a matter of concern. In 20 knees of 10 goats, we created a standardised donor defect in the knee. In the control group the defects were left empty, whereas in two other groups a bone plug from the proximal tibia was press-fitted into the defect with or without a covering periosteal layer. Histological evaluation after 8 and 16 weeks showed that relatively rapid osteoclastic resorption of the bone plug occurred. Defects were mainly filled with fibrous tissue, and collapse of the adjacent bone and cartilage was visible, especially when the defects were left empty. Occasionally, some consolidation of the graft to the host bone could be detected, and in some samples periosteal chondrogenesis was present. Our findings suggest that transplantation of a tibial bone plug with covering periosteum to the donor defect in a cartilage transplantation procedure does not have any additional value in an attempt to minimise the damage at the donor site. The observed resorption of the bone plug and the collapse of the adjacent joint margin remain a matter of concern with this technique.

Combined effects of insulin-like growth factor-1 and transforming growth factor-β1 on periosteal mesenchymal cells during chondrogenesis in vitro

Objective Periosteum contains undifferentiated mesenchymal stem cells that have both chondrogenic and osteogenic potential, and has been used to repair articular cartilage defects. During this process, the role of growth factors that stimulate the periosteal mesenchymal cells toward chondrogenesis to regenerate articular cartilage and maintain its phenotype is not yet fully understood. In this study, we examined the effects of insulin-like growth factor-1 (IGF-1) and transforming growth factor-β1 (TGF-β1), alone and in combination, on periosteal chondrogenesis using an in vitro organ culture model.Methods Periosteal explants from the medial proximal tibia of 2-month-old rabbits were cultured in agarose under serum free conditions for up to 6 weeks. After culture the explants were weighed, assayed for cartilage production via Safranin O staining and histomorphometry, assessed for proliferation via proliferative cell nuclear antigen (PCNA) immunostaining, and assessed for type II collagen mRNA expression via in situ hybridization.Results IGF-1 significantly increased chondrogenesis in a dose-dependent manner when administered continuously throughout the culture period. Continuous IGF-1, in combination with TGF-β1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-β1 gave the strongest proliferative stimulus early during chondrogenesis. In situ hybridization for type II collagen showed that continuous IGF-1 maintained type II collagen mRNA expression throughout the cambium layer from 2 to 6 weeks.Conclusion The results of this study demonstrate that IGF-1 and TGF-β1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis. Copyright 2003 OsteoArthritis Research Society International. Published by Elsevier Science Ltd. All rights reserved.

Induction of CD-RAP mRNA during periosteal chondrogenesis

Induction of chondrogenesis and maintenance of the chondrocyte phenotype are critical events for autologous periosteal transplantation, which is a viable approach for cartilage repair. Cartilage-derived retinoic acid-sensitive protein (CD-RAP) is a recently discovered protein that is mainly produced in cartilage. During development, CD-RAP expression starts at the beginning of chondrogenesis and continues throughout cartilage maturation. In order to investigate the involvement of CD-RAP during periosteal chondrogenesis we have determined the nucleotide sequence of the rabbit CD-RAP mRNA and utilized this information to evaluate the temporal and spatial expression pattern of CD-RAP at the mRNA level during chondrogenesis. When the periosteal explants were cultured under chondrogenic conditions, the expression of CD-RAP was induced, as shown by a 40-fold increase in CD-RAP mRNA between days 7 and 10. The temporal expression pattern of CD-RAP closely mimicked that of collagen type IIB mRNA. Also, the CD-RAP mRNA was localized to the matrix forming chondrocytes in the cambium layer of the periosteum by in situ hybridization as indicated by colocalization with collagen type II mRNA and positive safranin O staining. These data suggest a regulatory role of CD-RAP in periosteal chondrogenesis, which is potentially important for both cartilage repair and fracture healing via callus formation.

Brief exposure to high-dose transforming growth factor-beta1 enhances periosteal chondrogenesis in vitro: a preliminary report.

Articular cartilage has limited potential for repair. There have been various attempts aimed at improving the repair process in articular cartilage. Transforming growth factor-beta1 (TGF-beta1) has a stimulatory effect on chondrogenesis in periosteal explants. The purpose of the present study was to determine the effect of brief exposures (i.e., thirty and sixty minutes) of high concentrations of TGF-beta1 on periosteal chondrogenesis. Five hundred and seventy-three periosteal explants were harvested from forty-six two-month-old male New Zealand White rabbits. Explants were exposed to 50 or 100 ng/mL of TGF-beta1 for thirty or sixty minutes. The amount of cartilage formed was then determined with use of a standardized six-week agarose culture assay. There was a significant increase in the amount of cartilage formation (p < 0.01), Type-II collagen content (p < 0.05), and sulfate incorporation (p < 0.0001) in explants treated with TGF-beta1. Maximal stimulation occurred following exposure to 100 ng/mL of TGF-beta1 for thirty minutes. There was also an increase in chondrocyte proliferation as measured by [ (3) H-] thymidine incorporation on day 5 of culture (p < 0.049). The findings of this study indicate that exposure to TGF-beta1 has a stimulatory effect on periosteal chondrogenesis. This stimulatory effect is observed even with a very brief exposure time of thirty minutes. A possible clinical application of these findings is exposure of periosteal grafts that are currently utilized clinically to resurface articular defects to TGF-beta1 during the short time between graft procurement and implantation into the joint. This may obviate the need for intra-articular administration of TGF-beta1 and may enhance the ultimate graft incorporation and quality of cartilage repair.

The spatiotemporal expression of TGF-β1 and its receptors during periosteal chondrogenesis in vitro

Transforming growth factor-β1 (TGF-β1) has been shown to stimulate chondrogenesis in periosteal explants cultured in agarose suspension. TGF-βs exert their cellular effects through a heteromeric cell membrane receptor complex consisting of TGF-β type I and type II receptors. In this study, the spatial and temporal expressions of the type I receptor (TβR-I), type II receptor (TβR-II) and endogenous TGF-β1 in periosteal explants cultured in vitro were examined using reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The temporal changes in the expression of the TβR-I and TβR-II mRNAs correlated with that of TGF-β1. Exogenous administration of TGF-β1 upregulated the expression of both receptors and of the TGF-β1 ligand in a biphasic pattern. The earlier peak of upregulation was observed at 7 days in culture. A later peak of upregulation was seen at 42 days, at which time cartilage formation reached a maximum. Immunohistochemical studies demonstrated co-localization of TβR-I and TβR-II simultaneously among the same cells expressing TGF-β1. TGF-β1 treatment increased the expression of TGF-β1, TβR-I and TβR-II in mesenchymal cells in the cambium layer at 7 days in culture. Small round chondrocytes showed widely distributed immunoreactivity of TGF-β1, TβR-I and TβR-II in the 42-day explants treated with TGF-β1. These observations support the hypothesis that TGF-β1 regulates the initiation and formation of cartilage during periosteal chondrogenesis.

Effects of different embedding gels on periostealchondrogenesis in vitro.

To develop a more useful organ culture model for periosteal chondrogenesis in vitro, we compared the effects of the embedding of explants in agarose versus collagen gels. Chondrogenic differentiation was examined by means of histological observation and in terms of the expression of mRNA encoding two cartilage markers, Type II collagen and Aggrecan. Periosteal explants were derived from the tibiae of rabbits. These explants were embedded in either agarose gel or collagen gel and cultured for 6 weeks. Histological examinations revealed that in the agarose gel, cells were neither present in the explants nor in the gel. In the collagen gel, cells migrated from the explanted tissues into the gel, and some cells were round. However, no explants showed safranin-O staining. Only 10% of 10 surviving explants in the agarose gel expressed the Type II collagen gene and the Aggrecan gene. On the other hand, of 25 surviving explants in the collagen gel, the expression of the Type II collagen gene was detected in 18 explants (72%) and that of the Aggrecan gene was detected in 21 explants (84%). In conclusion, we demonstrated that the periosteum exerts chondrogenesis in certain circumstances and its chondrogenesis is closely related to the culture material.

Expression of β1 integrins during periosteal chondrogenesis

Objective The interactions between integrins and extracellular matrix proteins are known to modulate cell behavior, and may be involved in regulating cartilage formation and repair. The purpose of this study was to determine the patterns and localization of expression of the β1 integrins during cartilage formation by periosteum, which is used to repair articular cartilage.Design Periosteal explants from 2-month-old rabbit medial proximal tibiae were cultured in agarose suspension for 0 to 6 weeks, with 10ng/ml transforming growth factor-β1 added for the first 2 days of culture. Integrin expressions were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and localized by immunohistochemistry.Results Normal periosteum expressed the α1, α3, α5, β1 subunits at low levels, and the proteins for all but the α3 subunits were identified by immunohistochemistry in the periosteum. Significant two- to five-fold up-regulation of the mRNA expression of the α1, α3, α5 and β1 integrin subunits during the early proliferative stage of chondrogenesis was observed. The initial change was a five-fold increase in α5 expression on day 2 and a two-fold increase in α3 expression. On day 5, α1 expression was up-regulated (four-fold). β1 expression was broadly up-regulated (three to four-fold) from day 5 to 14. In the early stage of chondrocyte differentiation, after day 14, α1 expression was down-regulated, while there was upregulation of α3 (three-fold), α5 (three-fold) and β1 (four-fold) expressions. Thereafter, α1 expression was down-regulated, while α3, α5 and β1 expressions were up-regulated again during matrix synthesis. Immunohistochemistry confirmed this late decrease in α1 levels and increase in α3, α5 and β1 levels in chondrocytes.Conclusions These observations indicate that the β1 integrins play an important role in the process of chondrogenesis in periosteum.

Temporal expression patterns of BMP receptors and collagen II (B) during periosteal chondrogenesis

Articular cartilage has a limited ability to repair itself. Periosteal grafts have chondrogenic potential and are used clinically to repair defects in articular cartilage. An organ culture model system for in vitro rabbit periosteal chondrogenesis has been established to study the molecular events of periosteal chondrogenesis in vitro. In this model, bone morphogenetic protein-2 (BMP2) mRNA expression was found to be upregulated in the first 12 h. BMPs usually transduce their signals through a receptor complex that includes type II and either type IA or type IB BMP receptors. Receptors IA and IB play distinct roles during limb development. We have examined the temporal expression patterns for the mRNAs of these receptors using our experimental model. The mRNA expression patterns of these three BMP receptors differed from one another in periosteal explants during chondrogenesis. When these explants were cultured under chondrogenic conditions (agarose suspension with TGF-β1 added to the media for the first 2 days), the expression of BMPRII mRNA and that of BMPRIA mRNA varied only slightly and persisted over a long time. In contrast, the expression of BMPRIB mRNAwas upregulated within 12 h, peaked at day 5, and fell to a level that was barely detected beyond day 21. Moreover, the expression of BMPRIB mRNA preceded that of collagen type IIB mRNAs, a marker for matrix-depositing chondrocytes. These data support a role for coordinate expression of BMP2 and its receptors early during periosteal chondrogenesis.