Culturing periosteum in vitro: the influence of different sizes of explants

Periosteal transplantation is being used clinically to repair articular defects. Isolated cells and very small periosteal explants can be grown in tissue culture, but it will be necessary to test larger sizes for tissue engineering to be applied to clinical transplantation of periosteum. This study was conducted to assess the chondrogenic potential of different sizes of periosteal explants in agarose culture. Ninety-six rabbit tibial periosteal explants in three different sizes (small 1.5 × 2, medium 3 × 2, and large 4 × 6 mm, 32 pieces per size) were cultured in agarose suspension for 6 wk and given TGF-β1 (10 ng/mL) for the first 2 wk. Tissue growth, as indicated by normalized final wet weights of the explants after 6 wk in culture, was inversely proportional to explant size. Cartilage formation was observed in all explants. Histomorphometry revealed that cartilage formation was significantly better for the smaller explants (80% cartilage), but similar in the medium and larger explants (60% cartilage). Similar proportions of type II collagen were present in the different-sized explants. This study demonstrates that various sizes of periosteal explants can be grown in culture. Abundant cartilage was produced even by the large explants.     

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

BACKGROUND: 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. METHODS: 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. RESULTS: 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). Conclusions: 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. Clinical Relevance: 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.     

Chondrocytes in culture produce a mechanically functional tissue

A mechanically testable tissue was grown in vitro from rabbit chondrocytes that were initially plated at high density (approximately 80.000 cells/cm²). The DNA, collagen, and proteoglycan content, as well as the tissue thickness, tensile stiffness, and synthesis rates, were measured at 4, 6, and 8 weeks. The biochemical properties were similar to those for immature cartilage, with predominantly type-II collagen produced; this indicated that the cells retained their chondrocytic phenotype. The tissue formed a coherent mechanical layer with testable tensile stiffness as early as 4 weeks. The tensile elastic modulus reached 1.3 MPa at 8 weeks, which is in the range of values for native cartilage from the midzone. Collagen density was approximately 24 mg/ml at 8 weeks, which is about one-half the value for native cartilage, and the collagen fibril diameters were smaller. Chondrocytes in culture responded to culture conditions and were stimulated by cytokine interleukin-1β. When culture conditions were varied to RPMI nutrient medium with lower fetal bovine serum and higher ascorbic acid concentrations, the thickness decreased and the modulus increased significantly. Interleukin-1β. added to the 8-week culture for 2 weeks. caused a decrease of 60% in thickness, a decrease of 81% in proteoglycan content, and a decrease of 31% in collagen content; this is similar to the response of cartilage explants to interleukin-1β. This cartilage analog may be useful as a model system to study structure-function relationships in cartilage or as cartilage-replacement tissue.     

Biosynthesis and processing of collagens in different cartilaginous tissues

The distribution, structure, and biosynthesis of various collagen types have been studied in growth and structural cartilage from young rabbits. The major collagen of cartilage is α1(II); however, all cartilage matrix also contains 1α, 2α, 3α (Type C_m), as well as a high molecular weight disulfidelinked collagen (Type M). Cartilage fragments in organ culture demonstrate synthesis of precursors of collagen α chains and processing to their final forms. Although Type C_m collagen is present in the same proportion in the matrix of growth and structural cartilage, in vitro radiolabeling of rabbit cartilage showed that only growth cartilage is capable of actively synthesizing Type C_m, except in the newborn period when synthesis of Type C_m does occur in structural cartilage. A low molecular weight collagen (designated G collagen) is synthesized in vitro by growth cartilage but not by structural or articular cartilage. Preferential distribution of these minor collagens in growth cartilage suggests a role in development during normal cartilage growth.     

Relationship of donor site to chondrogenic potential of periosteum in vitro

Periosteum has been shown in vitro and in vivo to have a chondrogenic potential that permits it to be used for cartilage regeneration. A useful donor site should have good chondrogenic potential, availability of a large quantity of periosteum, and relative ease of access, and it should be associated with a low rate of morbidity. We hypothesized that the chondrogenic potential of periosteum varies from one bone to another and among different regions of the periosteum from a single bone. A total of 370 periosteal and 37 fascia lata (control) explants were taken from the skull, the ilium, the scapula, the upper, middle, and lower medial proximal tibia, the posterior proximal tibia, and the distal tibia of 2-month-old New Zealand rabbits. The explants were cultured for 6 weeks in agarose/Dulbecco's modified Eagle medium to which 10 ng/ml of transforming growth factor-β1 was added during the first 2 weeks. Skeletal muscle and fascia lata were used as controls. In addition, the thickness, cell density, and total cell count of the cambium layer were measured in 24 explants from the donor sites on the ilium and the upper, middle, and lower proximal tibia. At 6 weeks, histomorphometry and quantitative collagen typing were performed. The periosteal donor sites could be grouped into three categories according to chondrogenic potential: ilium (best), scapula and tibia, and skull (no chondrogenesis). The scapular periosteum was slightly better than that from the tibia. Within the tibia, the upper and middle zones of the proximal region were similar and were slightly better than the lower proximal tibia or the distal tibia. The cellularity of the cambium layer correlated positively with the amount of cartilage as a percentage of the total area. The results of this study indicate that iliac periosteum exhibited the best overall chondrogenic potential in vitro but that periosteum from the traditionally used medial proximal tibia also was excellent. Periosteum from the skull was not chondrogenic. The chondrogenic potential of periosteum varies from bone to bone and within the periosteum from one bone. This variation in chondrogenic potential among donor sites may be due to a difference in the total cell count of the cambium layer.     

Combined effects of insulin-like growth factor-1 and transforming growth factor-beta1 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-beta1 (TGF-beta1), 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-beta1 for the first 2 days, further enhanced overall total cartilage growth. Immunohistochemistry for PCNA revealed that combining IGF-1 with TGF-beta1 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-beta1 can act in combination to regulate proliferation and differentiation of periosteal mesenchymal cells during chondrogenesis.     

Co-Culture of Adipose Derived Stem Cells and Chondrocytes with Surface Modifying Proteins Induces Enhanced Cartilage Tissue Formation

ABSTRACT

Objectives: Current treatments for focal cartilage defects include osteochondral allograft transplants—a common treatment for large defects and revisions of previously autografted joints. Allografts with weak osseous regions are usable, since bone remodeling replaces inferior quality bone. However, poor quality chondral surfaces on grafts preclude their use, leading to grafting material shortages. Endogenous adult stem cells can make hyaline-like cartilage tissue on scaffolds. To increase the number of usable allografts, tissue culture methods using adipose derived stem cells (ASCs) were developed to grow cartilage on grafts. Methods: Co-cultures utilized living chondrocytes in host cartilage, modeling in vivo conditions, and ASCs seeded on the allografts. Sterilized allografts were treated with Poly-L-Lysine and ProNectin. Tissue growth was analyzed and quantified with histological techniques. Results and Conclusions: Monoculture experiments produced tenuous cartilage formation when proteins were utilized and allograft surfaces were perforated. Extensive tissue formation was observed with co-culture and the presence of type II collagen was confirmed with immunohistochemistry. Results demonstrate that co-culture techniques offer a better means of growing tissue on allograft cartilage surfaces. Additionally, the use of proteins to facilitate surface attachment produced more tissue formation demonstrating that cell attachment is crucial when growing cartilage on allografts. Development of new culture techniques to evaluate treatment strategies will accelerate the rate at which cartilage procedures using endogenous cells are possible. This will increase the number of usable grafts and allow critical selection of grafts to fit specific surfaces increasing surgical success by returning the joint to its native structure.     

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 microg/ml growth hormone), and/or ITS+ (2.08 microg/ml each of insulin, transferrin, and selenious acid, plus 1.78 microg/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 microg/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.     

Integrative repair of articular cartilage in vitro: Adhesive strength of the interface region

The objective of this study was to quantify the strength of the repair tissue that forms at the interface between pairs of cartilage explants maintained in apposition in an in vitro culture system. Articular cartilage explants were harvested from calves and from adult bovine animals, dissected into uniform blocks, and incubated in pairs within a chamber that maintained a 4 × 5 mm area of tissue overlap. Following 1–3 weeks of incubation, integrative repair was assessed by testing samples in a tensile single-lap configuration to estimate adhesive strength. After incubation in medium containing 20% fetal bovine serum, the adhesive strength between pairs of calf cartilage blocks and pairs of adult bovine cartilage blocks increased at a rate of 7.0 and 10.5 kPa/week, respectively. This repair process appeared to be dependent on viable cells, since lyophilization of adult bovine cartilage before incubation completely inhibited the development of an interface with a measurable adhesive strength. The repair process was dependent on serum components in the medium. Incubation of sample pairs for 3 weeks in medium supplemented with 20% fetal bovine serum resulted in a relatively high proteoglycan content as well as a relatively high adhesive strength (34 kPa), whereas incubation in basal medium with or without 0.1% bovine serum albumin resulted in a 54–70% lower proteoglycan content and a 65–88% lower adhesive strength. Samples incubated for 3 weeks with serum also had a 20% higher DNA content than samples maintained in basal medium. Histological analysis indicated some cell division at the free surfaces of the explant and also occasional cells within the interface region between explants.     

The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure.

Cartilage repair by autologous periosteal arthroplasty is enhanced by continuous passive motion (CPM) of the joint after transplantation of the periosteal graft. However, the mechanisms by which CPM stimulate chondrogenesis are unknown. Based on the observation that an oscillating intra-synovial pressure fluctuation has been reported to occur during CPM (0.6-10 kPa), it was hypothesized that the oscillating pressure experienced by the periosteal graft as a result of CPM has a beneficial effect on the chondrogenic response of the graft. We have developed an in vitro model with which dynamic fluid pressures (DFP) that mimic those during CPM can be applied to periosteal explants while they are cultured in agarose gel suspension. In this study periosteal explants were treated with or without DFP during suspension culture in agarose, which is conducive to chondrogenesis. Different DFP application times (30 min, 4 h, 24 h/day) and pressure magnitudes (13, 103 kPa or stepwise 13 to 54 to 103 kPa) were compared for their effects on periosteal chondrogenesis. Low levels of DFP (13 kPa at 0.3 Hz) significantly enhanced chondrogenesis over controls (34 +/- 7% vs 14 +/- 5%; P < 0.05), while higher pressures (103 kPa at 0.3 Hz) completely inhibited chondrogenesis, as determined from the percentage of tissue that was determined to be cartilage by histomorphometry. Application of low levels of DFP to periosteal explants also resulted in significantly increased concentrations of Collagen Type II protein (43 +/- 8% vs 10 +/- 5%; P < 0.05). New proteoglycan synthesis, as measured by 35S-sulphate uptake was increased by 30% in periosteal explants stimulated with DFP (350 +/- 50 DPM vs 250 +/- 75 DPM of 35S-sulphate uptake/microg total protein), when compared to controls though this difference was not statistically significant. The DFP effect at low levels was dose-dependant for time of application as well, with 4 h/day stimulation causing significantly higher chondrogenesis than just 30 min/day (34 +/- 7 vs 12 +/- 4% cartilage; P < 0.05) and not significantly less than that obtained with 24 h/day of DFP (48 +/- 9% cartilage, P > 0.05). These observations may partially explain the beneficial effect on cartilage repair by CPM. They also validate an in vitro model permitting studies aimed at elucidating the mechanisms of action of mechanical factors regulating chondrogenesis. The fact that these tissues were successfully cultured in a mechanical environment for six weeks makes it possible to study the actions of mechanical factors on the entire chondrogenic pathway, from induction to maturation. Finally, these data support the theoretical predictions regarding the role of hydrostatic compression in fracture healing.     

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.     

Damage and degenerative changes in menisci‐covered and exposed tibial osteochondral regions after simulated landing impact compression—a porcine study

The capacity of menisci‐covered and exposed tibial osteochondral regions in resisting impact‐induced damage and degeneration is not fully understood. This study sought to evaluate damage and degenerative changes in these regions upon a single simulated landing impact. We hypothesized that the menisci‐covered regions are more susceptible to damage and degeneration than their exposed counterparts. Menisci‐covered and exposed tibial osteochondral explants were extracted from fresh porcine hind legs and placed in culture up to 14 days. Impact compression, based on a single 10‐Hz haversine, was performed at Day 1. Control (non‐impact) and impacted explants were randomly selected for cell viability assessment, glycoaminoglycan and collagen content assays, histology, immunohistochemistry, and micro‐computed tomography. When subjected to 2‐mm displacement compression, exposed explants achieved a significantly higher peak impact stress (p < 0.05) than menisci‐covered explants. No significant difference in cell viability, glycoaminoglycan and collagen content, and Mankin scores (p > 0.05) was observed between both explant groups. Both groups were observed with reduced proteoglycan and type II collagen staining at Day 14; the exposed group was noted with increased cartilage volume at Days 7–14. The inferior resistance of menisci‐covered regions, against impact‐induced damage and degeneration, is a potential factor that may contribute to the meniscectomy model of osteoarthritis. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 1100–1108, 2009     

Sustained hypoxia enhances chondrocyte matrix synthesis

Articular cartilage is an avascular tissue with chondrocytes in the deeper zones existing under conditions of sustained hypoxia. Using a hypoxic chamber to provide controlled hypoxia, this study was performed to determine whether sustained hypoxia enhances the production of cartilage matrix proteins. Freshly isolated primary bovine articular chondrocytes were encapsulated in three-dimensional alginate beads and maintained at 2% oxygen with media changes using media pre-equilibrated to 2% oxygen. Immunolocalization of HIF-1α was performed to verify hypoxic conditions. Sustained hypoxia resulted in an increase in proteoglycan synthesis after only 1 day, as measured by ³⁵S-sulfate incorporation. This increase was maintained for the duration of the 17 day study. After 17 days of hypoxic culture, increases in total type II collagen and COL2A1 gene expression were probed by indirect immunofluorescence, type II collagen ELISA, and real-time qPCR; in addition, increased glycosaminoglycan deposition was observed as determined by chemical analysis. These studies show that sustained hypoxia enhances articular chondrocyte matrix synthesis and viability in three-dimensional alginate culture. © 2008 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 27: 793–799, 2009     

The chondrogenic potential of periosteum decreases with age.

Periosteum contains undifferentiated mesenchymal stem cells that possess the potential for chondrogenesis during cartilage repair and in fracture healing. With aging, the chondrogenic potential of periosteum declines significantly. An organ-culture model was used to investigate the relationship between the chondrogenic potential of periosteum and aging. A total of 736 periosteal explants from the proximal medial tibiae of 82 rabbits, aged 2 weeks to 2 years, were cultured in agarose suspension conditions conductive for chondrogenesis. and analyzed using histomorphometry, collagen typing, wet weight measurement, 3H-thymidine and 35S-sulfate uptake, autoradiography, and PCNA immunostaining. The rabbits were skeletally mature by 6 months and stopped increasing in weight by 12 months. Chondrogenesis declined significantly with age (P < 0.0001) and was maximal in the 1.5-2 month-old rabbits. Explants from the 6 month-old rabbits formed 50% less cartilage. and by 12 months chondrogenesis reached a steady state minimal level. In parallel with this decrease in chondrogenic potential similar decreases were measured in 3H-thymidine uptake (P < 0.0001). 35S-sulfate uptake (P = 0.0117), as well as the thickness (P < 0.0001) and the total number of cells in the cambium layer of the periosteum (P < 0.0001). Autoradiography with 3H-thymidine and PCNA immunostaining confirmed the measured decrease in proliferative activity in the cambium layer where the chondrocyte precursors reside, although the percentage of proliferating cells did not change significantly with age. The most dramatic change was the marked decrease (87%) in the thickness and total cell number in the cambium layer of the perisoteum between the 2 and 12 month-old rabbits (P < 0.05). These data confirm a decline in the chondrogenic potential of periosteum with aging. Thus, one possibility for improving cartilage formation by periosteal transplantation after skeletal maturity would be to stimulate an increase in the total number of cells in the chondrocyte precursor pool early during chondrogenesis.     

Tissue neogenesis and STRO‐1 expression in immature and mature articular cartilage

This study determined the potential for neotissue formation and the role of STRO‐1+ cells in immature versus mature articular cartilage. Cartilage explants from immature and mature bovine knee joints were cultured for up to 12 weeks and stained with safranin‐O, for type II collagen and STRO‐1. Bovine chondrocyte pellet cultures and murine knee joints at the age of 2 weeks and 3 months, and surgically injured cartilage, were analyzed for changes in STRO‐1 expression patterns. Results show that immature explants contained more STRO‐1+ cells than mature explants. After 8 weeks in culture, immature explants showed STRO‐1+ cell proliferation and newly formed tissue, which contained glycosaminoglycan and type II collagen. Mature cartilage explants showed only minimal cell expansion and neotissue formation. Pellet cultures with chondrocytes from immature cartilage showed increased glycosaminoglycan synthesis and STRO‐1+ staining, as compared to pellets with mature chondrocytes. The frequency of STRO‐1+ cells in murine knee joints significantly declined with joint maturation. Following surgical injury, immature explants had higher potential for tissue repair than mature explants. In conclusion, these findings suggest that the high percentage of STRO‐1+ cells in immature cartilage changes with joint maturation. STRO‐1+ cells have the potential to form new cartilage spontaneously and after tissue injury. © 2009 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:96–102, 2010     

Differences in the secretome of cartilage explants and cultured chondrocytes unveiled by SILAC technology

The main goal of our study was to analyze and compare the profiles of secreted proteins from adult human articular chondrocytes in monolayers, and cartilage explants in culture, using a de novo protein labeling approach. Stable isotope labeling of proteins in culture was used to differentiate between chondrocyte‐derived proteins and other preexisting matrix‐derived components, or proteins coming from serum or synovial fluids. Proteins in culture supernatants were resolved by one‐dimensional SDS‐PAGE electrophoresis, and analyzed in tandem with LC/MS‐MS (liquid chromatography/double mass spectrometry). Results from stable isotope labeling with amino acids in cell culture (SILAC) were validated by specific immunoblotting of four relevant proteins identified in the secretion media. After 8–10 days of culture, over 90% of proteins secreted during monolayer growth contained ¹³C₆‐Arg and ¹³C₆‐Lys. Nonlabeled proteins corresponded mostly to plasma‐associated proteins, indicating background contamination of medium with serum remnants. The majority of the secreted proteins in 2D cultures were extracellular matrix components and matrix regulators, along with some inflammatory agents and metabolic enzymes. In explants, only 25%–30% of proteins were labeled with heavy amino acids, corresponding to matrix regulators and carrier molecules. Nonlabeled proteins corresponded primarily to structural matrix components. In qualitative terms, all labeled proteins coming from cartilage explants were also found in chondrocytes supernatants. In summary, our results show differences in the labeling pattern of proteins found in supernatants from explants and monolayers. Most proteins found in the media of explants were subproducts of matrix turnover rather than newly synthesized. To our knowledge, this study is the first one so far applying SILAC technology in the context of cartilage and chondrocytes physiology. © 2010 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 28:1040–1049, 2010     

Avian medullary bone in organ culture: Effects of vitamin D metabolites on collagen synthesis

Summary A new organ culture system for the study of bone metabolism has been developed using chicken medullary bone. The presence of viable bone cells in culture was demonstrated by histological and histochemical techniques. Incorporation of³H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP) was determined using purified bacterial collagenase. Collagen accounted for approximately 10–15% of the total protein labeled. The addition of 1,25-dihydroxycholecalciferol (1,25 (OH)₂D₃) resulted in a dose-dependent inhibition of³H-proline incorporation into CDP at doses from 10⁻¹⁰M to 10⁻⁷M, with maximal suppression reaching 30% of control. The effect was specific for collagen, since³H-proline incorporation into NCP was unaffected. Hydroxyproline analysis of bone explants and culture medium revealed a 1,25(OH)₂D₃-induced decrease in the³H-hydroxyproline content of the system (bone + medium), suggesting that the effect of 1,25(OH)₂D₃ is due to inhibition of collagen synthesis rather than enhanced collagen degradation, impaiored incorporation of collagen into bone matrix, or bone resorption Medullary bone collagen synthesis was not affected by 24,25(OH)₂D₃, either alone or in combination with 1,25(OH)₂D₃. Structure-activity studies of vitamin D metabolites showed that 1,25(OH)₂D₃ and 1,24,25(OH)₃D₃ were the most potent metabolites tested, followed by 1-alpha(OH)D₃. 25(OH)D₃ and 24,25(OH)₂D₃ had no effect at concentrations as high as 10⁻⁷M. These results indicate a possible role for vitamin D in the regulation of medullary bone formation during the reproductive cycle of the egg-laying hen, and suggest the potential utility of medullary bone as anin vitro model for the study of bone formation     

Gene and Protein Expression During Differentiation and Matrix Mineralization in a Chondrocyte Cell Culture System

Endochondral bone formation occurs through a series of developmentally regulated cellular stages, from initial formation of cartilage tissue to calcified cartilage, resorption, and replacement by bone tissue. Nasal cartilage cells isolated by enzymatic digestion from rat fetuses were seeded at a final density of 10⁵ cell/cm² and cultured in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum in the presence of ascorbic acid and β-glycerophosphate. First, cells lost their phenotype but in this condition they rapidly reexpressed the chondrocyte phenotype and were able to form calcified cartilaginous nodules with the morphological appearance of cartilage mineralization that occurs in vivo during endochondral ossification. In this mineralizing chondrocyte culture system, we investigated, between day 3 and day 15, the pattern expression of types II and X collagen, proteoglycan core protein, characteristic markers of chondrocyte differentiation, as well as alkaline phosphatase and osteocalcin associated with the mineralization process. Analysis of labeled collagen and immunoblotting revealed type I collagen synthesis associated with the loss of chondrocyte phenotype at the beginning of the culture. However, our culture conditions promoted extracellular matrix mineralization and cell differentiation towards the hypertrophic phenotype. This differentiation process was characterized by the induction of type X collagen mRNA, alkaline phosphatase, and diminished expression of type II collagen and core protein of large proteoglycan after an increase in their mRNA levels before the mineralizing process. These results revealed distinct switches of the specific molecular markers and indicated a similar temporal expression to that observed in vivo recapitulating all stages of the differentiation program in vitro. Received: 12 December 1996 / Accepted: 26 June 1997     

The effect of recombinant human fibroblast growth factor‐18 on articular cartilage following single impact load

The aim of this in vitro study was to ascertain the effect of recombinant human Fibroblast Growth Factor‐18 (rhFGF18) on the repair response of mechanically damaged articular cartilage. Articular cartilage discs were harvested from healthy mature horses (n = 4) and subjected to single impact load (SIL). The impacted explants, together with unimpacted controls were cultured in modified DMEM ± 200 ng/ml rhFGF18 for up to 30 days. Glycosaminoglycan (GAG) release into the media was measured using the dimethylmethylene blue (DMMB) assay. Aggrecan neopepitope CS846, collagen type II synthesis (CPII) and cleavage (C2C) were measured by ELISA. Histological analysis and TUNEL staining were used to assess repair cell number and cell death. Impacted explants treated with rhFGF18 showed significantly more GAG and CS846 release into the media (p < 0.05), there was also a significant decrease in C2C levels at Day 20. Loaded sections treated with rhFGF18 had more repair cells and significantly less cell death (p < 0.001) at Day 30 in culture. In an in vitro damage/repair model, rhFGF18 increases the proteoglycan synthesis, the repair cell number and prevents apoptosis at Day 30. This suggests that rhFGF18 may be a good candidate for enhancement of cartilage repair following mechanical damage. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 32:923–927, 2014.