Recombinant Human Transforming Growth Factor-beta Research Articles

Enhancing the production of recombinant human TGF-β1 through an understanding of TGF-β1 synthesis, signaling, and endocytosis in CHO cells.

To enhance the production of recombinant human transforming growth factor-beta1 (rhTGF-β1) in Chinese hamster ovary (CHO) cells, rhTGF-β1 was first characterized for endocytosis, signaling pathway, and overall maturation process. The mature rhTGF-β1 used for clinical application was internalized into CHO cells and inhibited the growth of CHO cells in a dose-dependent manner. However, mature rhTGF-β1 was mostly produced in the form of latent rhTGF-β1 in cultures of recombinant CHO (rCHO) cells producing rhTGF-β1 (CHO-rhTGF-β1). The concentration of active mature rhTGF-β1 in the culture supernatant of CHO-rhTGF-β1 cells was not high enough to compromise yield. In addition, a significant amount of unprocessed precursors was produced by CHO-rhTGF-β1 cells. Overexpression of PACEsol, a soluble form of furin, in CHO-rhTGF-β1 cells was effective for the proteolytic cleavage of unprocessed precursors. The highest mature rhTGF-β1 concentration (6.4μgmL-1 ) was obtained with the PACEsol-expressing clone, which was approximately 45% higher than that of the parental clone (P<0.01). Thus, a comprehensive understanding of the intrinsic properties of rhTGF-β1 with respect to the overall maturation process, signaling pathway, and endocytosis is essential for effectively enhancing the production of mature rhTGF-β1 in CHO cells.

Osteoblastic Differentiation of Human Stem Cells Derived from Bone Marrow and Periodontal Ligament Under the Effect of Enamel Matrix Derivative and Transforming Growth Factor-Beta

To increase the understanding of the applicability of biomaterials and growth factors in enhancing stem cell-based bone regeneration modalities, this study evaluated the effects of enamel matrix derivative (EMD) and recombinant human transforming growth factor-beta (rhTGF-β) on osteoblastic differentiation of human bone marrow mesenchymal stem cells (hBMSCs) as well as human periodontal ligament stem cells (hPDLSCs). hBMSCs and hPDLSCs were obtained, and identification of stem cell surface markers was performed according to the criteria of the International Society for Cellular Therapy. Each group of stem cells was separately treated with a serial dilution of EMD (10, 50, and 100 μg/mL) or rhTGF-β (10 ng/mL). Osteoblastic differentiation was examined through in vitro matrix mineralization by alizarin red staining, and mRNA expression of osteopontin and osteonectin was determined by quantitative reverse-transcriptase polymerase chain reaction. hPDLSCs were further assessed for osteocalcin mRNA expression. Stem cells cultured in osteogenic medium were employed as a standard positive control group. In none of the experimental groups were bone-related mRNAs detected subsequent to treatment with EMD for 5, 10, and 15 days. Alizarin red staining on day 21 was negative in EMD-treated BMSC and PDLSC cultures. In rhTGF-β-supplemented BMSC culture, expression of osteonectin mRNA was demonstrated on day 15, which was statistically comparable to the positive control group. Nevertheless, extracellular matrix mineralization was inhibited in both groups of stem cells. Within the limitations of this study, it could be concluded that EMD with a concentration of 10, 50, or 100 μg/mL has no appreciable effect on osteoblastic differentiation of BMSCs and PDLSCs. Application of rhTGF-β increased osteonectin mRNA expression in BMSCs. This finding corroborates the hypothesis that TGF-β might be involved in early osteoblastic maturation.

Effect of Bioactive Ceramic and Recombinant Human Transforming Growth Factor-Beta (rhTGF-beta) on Regeneration of Parietal Bone Defects in Rat

The purpose of this study was to evaluate the regenerative response of parietal bone defects in rats to bioactive ceramic and TGF-beta 1. Sixty mature adult rats with initial weights of 300 g to 324 g were used. The diameter of each defect was 3 mm. The animals were divided into four study groups consisting of 15 rats per group. Groups 1 and 3 (control groups) received 30 μg bioceramic per surgical defect site. Groups 2 and 4 (experimental groups) received 30 μg bioceramic combined with 10 μL of reconstituted TGF-beta 1 (200 ng). Animals of Groups 1 and 3 were euthanized at 18 days; Group 2 and 4 animals were terminated at 49 days after surgery. Each of the outcomes was analyzed via two-way analysis of variance model (ANOVA). Histologic reactions to the implants were similar with no significant difference between bioceramic and bioceramic + TGF-beta 1 specimens. In the 18-day specimens, the percentage of ceramic was marginally higher than the 49-day specimens. No significant difference was noted in the percentage of bone in 18-day and 49-day specimens. Based on the findings of this study, it was concluded that TGF-beta 1 promoted no significant increase in new bone when compared to bioactive ceramic alone.

Effect of rhTGF-β1 combined with bone grafts on human periodontal cell differentiation

Various techniques and materials have been proposed for the treatment of periodontal defects. In periodontal regeneration, periodontal ligament (PDL) cell differentiation as well as certain growth factors and their delivery system applied are critical. The purpose of this study was to evaluate the in vitro effect of recombinant human transforming growth factor-beta 1 (rhTGF-β1) combined with two different bone grafts on human PDL (hPDL) cell differentiation. The hPDL cells were treated with TGF-β1 alone or in combination with a calcified freeze-dried bone allograft (FDBA) and a porous biphasic calcium phosphate (BC) bone graft. Cell differentiation effect was estimated by measuring alkaline phosphatase (ALPase) activity and osteocalcin secretion. Results demonstrated that rhTGF-β1 alone or in combination with FDBA and BC provoked a significant (p < 0.05) increase in ALPase activity as compared with controls. The findings of this study confirmed the beneficial role of rhTGF-β1 combined with FDBA and BC as carriers in periodontal regeneration.

Induction of bone formation by transforming growth factor‐β2 in the non‐human primate Papio ursinus and its modulation by skeletal muscle responding stem cells

Four adult non-human primates Papio ursinus were used to study induction of bone formation by recombinant human transforming growth factor-beta(2) (hTGF-beta(2)) together with muscle-derived stem cells. The hTGF-beta(2) was implanted in rectus abdominis muscles and in calvarial defects with and without addition of morcellized fragments of striated muscle, harvested from the rectus abdominis or temporalis muscles. Expression of osteogenic markers including osteogenic protein-1, bone morphogenetic protein-3 and type IV collagen mRNAs from generated specimens was examined by Northern blot analysis. Heterotopic intramuscular implantation of 5 and 25 microg hTGF-beta(2) combined with 100 mg of insoluble collagenous bone matrix yielded large corticalized mineralized ossicles by day 30 with remodelling and induction of haematopoietic marrow by day 90. Addition of morcellized rectus abdominis muscle to calvarial implants enhanced induction of bone formation significantly by day 90. In Papio ursinus, in marked contrast to rodents and lagomorphs, hTGF-beta(2) induced large corticalized and vascularized ossicles by day 30 after implantation into the rectus abdominis muscle. This striated muscle contains responding stem cells that enhance the bone induction cascade of hTGF-beta(2). Induction of bone formation by hTGF-beta(2) in the non-human primate Papio ursinus may occur as a result of expression of bone morphogenetic proteins on heterotopic implantation of hTGF-beta(2); the bone induction cascade initiated by mammalian TGF-beta proteins in Papio ursinus needs to be re-evaluated for novel molecular therapeutics for induction of bone formation in clinical contexts.

A novel collagen scaffold supports human osteogenesis—applications for bone tissue engineering

Collagen glycosaminoglycan (CG) scaffolds have been clinically approved as an application for skin regeneration. The goal of this study has been to examine whether a CG scaffold is a suitable biomaterial for generating human bone tissue. Specifically, we have asked the following questions: (1) can the scaffold support human osteoblast growth and differentiation and (2) how might recombinant human transforming growth factor-beta (TGF-beta(1)) enhance long-term in vitro bone formation? We show human osteoblast attachment, infiltration and uniform distribution throughout the construct, reaching the centre within 14 days of seeding. We have identified the fully differentiated osteoblast phenotype categorised by the temporal expression of alkaline phosphatase, collagen type 1, osteonectin, bone sialo protein, biglycan and osteocalcin. Mineralised bone formation has been identified at 35 days post-seeding by using von Kossa and Alizarin S Red staining. Both gene expression and mineral staining suggest the benefit of introducing an initial high treatment of TGF-beta(1) (10 ng/ml) followed by a low continuous treatment (0.2 ng/ml) to enhance human osteogenesis on the scaffold. Osteogenesis coincides with a reduction in scaffold size and shape (up to 70% that of original). A notable finding is core degradation at the centre of the tissue-engineered construct after 49 days of culture. This is not observed at earlier time points. Therefore, a maximum of 35 days in culture is appropriate for in vitro studies of these scaffolds. We conclude that the CG scaffold shows excellent potential as a biomaterial for human bone tissue engineering.

Periodontal tissue regeneration by recombinant human transforming growth factor‐β3 in Papio ursinus

Osteogenic proteins of the transforming growth factor-beta superfamily induce periodontal tissue regeneration in animal models, including primates. To our knowledge, no studies have been performed in periodontal regeneration using the transforming growth factor-beta 3 isoform. In the present study, recombinant human transforming growth factor-beta 3 was examined for its ability to induce periodontal tissue regeneration in the nonhuman primate, Papio ursinus. Class II furcation defects were surgically created bilaterally in the maxillary and mandibular molars of four adult baboons. Heterotopic ossicles, for transplantation to selected furcation defects, were induced within the rectus abdominis muscle by recombinant human transforming growth factor-beta 3. Forty days later, the periodontal defects were implanted with recombinant human transforming growth factor-beta 3 in Matrigel as the delivery system, with recombinant human transforming growth factor-beta 3 plus minced muscle tissue in Matrigel, or with the harvested recombinant human transforming growth factor-beta 3-induced ossicles. Sixty days after periodontal implantation, the animals were killed and the specimens harvested. Histological analysis on undecalcified sections measured the area and volume of new alveolar bone and the coronal extension of newly formed alveolar bone and cementum. Morphometric analyses showed pronounced periodontal regeneration in experimental defects compared with controls. Substantial regeneration was observed in defects implanted with fragments of heterotopically induced ossicles and with recombinant human transforming growth factor-beta 3 plus minced muscle tissue. Recombinant human transforming growth factor-beta 3 in Matrigel significantly enhanced periodontal tissue regeneration in the nonhuman primate, P. ursinus.

Transforming Growth Factor-β1 Accelerates Resorption of a Calcium Carbonate Biomaterial in Periodontal Defects.

In a previous study, recombinant human transforming growth factor-beta1 (rhTGF-β1 ) in a calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for guided tissue regeneration (GTR) to study whether rhTGF-β1 would enhance or accelerate periodontal regeneration. The results showed minimal benefits of rhTGF-β1 , and a clear account for this could not be offered. One potential cause may be that the rhTGF-β1 formulation was biologically inactive. Several growth or differentiation factors have been suggested to accelerate degradation of biomaterials used as carriers. The objective of this study was to evaluate possible activity of rhTGF-β1 on biodegradation of the calcium carbonate carrier. rhTGF-β1 in a putty-formulated particulate calcium carbonate carrier was implanted into critical-size, supraalveolar periodontal defects under conditions for GTR in five beagle dogs. Contralateral defects received the calcium carbonate carrier combined with GTR without rhTGF-β1 (control). The animals were euthanized at week 4 post-surgery and block biopsies of the defect sites were collected for histologic and histometric analysis. Radiographs were obtained at defect creation and weeks 2 and 4 after defect creation. No statistically significant differences were observed in new bone formation (bone height and area) among the treatments. However, total residual carrier was significantly reduced in sites receiving rhTGF-β1 compared to control (P = 0.04). Similarly, carrier density was considerably reduced in sites receiving rhTGF-β1 compared to control; the difference was borderline statistically significant (P = 0.06). Within the limitations of the study, it may be concluded that rhTGF-β1 accelerates biodegradation of a particulate calcium carbonate biomaterial, indicating a biologic activity of the rhTGF-β1 formulation apparently not encompassing enhanced or accelerated periodontal regeneration.

Additive Enhancement of Implant Fixation Following Combined Treatment with rhTGF- 2 and rhBMP-2 in a Canine Model

Gaps at the interface between implant and bone increase the risk of diminished implant fixation and eventual loosening. The purpose of the present study was to determine if combined use of recombinant human transforming growth factor-beta 2 (rhTGF-beta2) and bone morphogenetic protein 2 (rhBMP-2) led to greater implant fixation strength in the presence of interface gaps than the use of either growth factor alone. Twenty-eight skeletally mature adult male dogs received one porous-coated titanium implant in the proximal part of each humerus, for a total of fifty-six implantation sites. Spacers were used to establish an initial 3-mm gap between the implant and the host bone at all fifty-six sites. Forty-two implants were coated with hydroxyapatite-tricalcium phosphate and were used in three growth-factor-treatment groups in which the implants placed in the left humerus were loaded with 12 microg of rhTGF-beta2 (Group 1, seven animals), 25 microg of rhBMP-2 (Group 2, seven animals), or 12 microg of rhTGF-beta2 combined with 25 microg of rhBMP-2 (Group 3, seven animals). In these animals, the twenty-one implants that were placed in the right humerus were loaded with buffer only to serve as contralateral controls. In Group 4 (seven animals), the implants were not coated with hydroxyapatite-tricalcium phosphate, the gap in the left humerus was lightly packed with autogenous bone graft, and the gap in the right humerus was left empty to serve as a contralateral control. All animals were killed at twenty-eight days. The primary end points included three mechanical variables: fixation strength, interface stiffness, and energy to failure. Secondary end points included bone ingrowth and bone volume and trabecular architecture in the gap and in a region located 2 mm medial to the implantation site. The hydroxyapatite-tricalcium phosphate coating had no effect on implant fixation, bone ingrowth, or bone formation in the 3-mm gap. Individual growth factor treatments led to 2.3 to 3.2-fold increases in fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.05). The combined growth factor treatment led to 5.7-fold increases in fixation strength and stiffness compared with the values for the contralateral controls (p < 0.01). Autogenous bone graft treatment was associated with 4.5 to 6.4-fold increases in implant fixation strength and stiffness as compared with the values for the contralateral controls (p < 0.01). Compared with the relevant contralateral controls, energy to failure was increased 3.5-fold in association with TGF-beta2 alone (p < 0.05), 4.5-fold in association with TGF-beta2 combined with BMP-2 (p < 0.01), and 2.5-fold in association with autogenous bone-grafting. As much as 63% of the variance in the mechanical end points was associated with variance in bone volume and architecture in the 3-mm gap and in the region of interest located 2 mm medial to the implantation site (p < 0.01). In this animal model, the combined use of TGF-beta2 and BMP-2 led to more secure mechanical fixation of the implant than did the use of either growth factor alone and demonstrated results that were similar to those associated with the use of autogenous bone graft.

Collagen-hydroxyapatite/tricalcium phosphate microspheres as a delivery system for recombinant human transforming growth factor-beta 1.

The purpose of this study is to evaluate the carrier capability of collagen-hydroxyapatite/tricalcium phosphate (Col-HA/TCP) microspheres to the rhTGF-beta 1 (recombinant human transforming growth factor-beta 1). After anesthesia, a bone defect (7.0 mm in diameter and 10.0 mm in depth) was created at the distal femoral condyles of New Zealand white rabbits. These defects were then completely filled with the implant materials. After 5, 7, 9, 11, 13, and 15 weeks, the animals were sacrificed and histological evaluations were performed. The results showed that when the defects were treated with Col-HA/TCP microspheres without rhTGF-beta 1, there was only spotty new bone formation during the 15 week experimental period and most of the defect was filled with fibrous tissue and inflammatory cells, whereas active bone formation with mature marrow tissue formation was evident in the defect treated with Col-HA/TCP microspheres containing rhTGF-beta 1. Collagen-hydroxyapatite/tricalcium phosphate microspheres were expected to be replaced by the regenerated bone structure as the bone reconstruction and bone-remodeling process occurred. It was apparent that bone regeneration was influenced by the addition of rhTGF-beta 1. Collagen-hydroxyapatite/tricalcium phosphate microspheres were a good carrier for rhTGF-beta 1.

Transforming growth factor-beta1 incorporated in calcium phosphate cement stimulates osteotransductivity in rat calvarial bone defects.

Bone regeneration of the alveolar crest around dental implants is an important factor in the success of implant use. Calcium phosphate cement can be used as a bone substitute and applied clinically as a paste to fill micro- and macroscopic bone defects. We have shown earlier that the intermixing of the recombinant human transforming growth factor-beta1 (rhTGF-beta1) in hardening calcium phosphate cement stimulated osteoblastic differentiation of rat primary bone cells in vitro. The aim of the present study was to examine whether the similar enrichment with rhTGF-beta1 affects the replacement of calcium phosphate cement by bone (osteotransduction) in calvarial critical size defects (csd) of adult rats. Two bone defects of 5 mm diameter were created bilaterally in each skull of 10 adult male rats. Both defects were filled with 53 mg of calcium phosphate cement without rhTGF-beta1 (control) at one side, and with 10 or 20 ng rhTGF-beta1 at the other side. After 8 weeks, defects with surrounding skull were analysed histologically and histomorphometrically. The addition of rhTGF-beta1 in the cement increased the amount of bone in rat skull defects. This finding coincidences with our in vitro observations, that intermixing of rhTGF-beta1 in calcium phosphate cement stimulates bone cell differentiation. Addition of rhTGF-beta1 stimulated bone formation as indicated by an increased bone volume of 50% and an increased bone/cement contact of 65%, in comparison to control defects with cement without rhTGF-beta1. In addition, rhTGF-beta1 reduced the remaining volume of cement, by 11% at 10 ng rhTGF-beta1, and by 20% at 20 ng rhTGF-beta1 in the cement. Defect closure was not affected. We conclude that the intermixing of rhTGF-beta1 in a fast-setting calcium phosphate cement stimulates bone growth and the osteotransduction of the cement. For bone regeneration procedures around endosseous implants, calcium phosphate cement with rhTGF-beta1 might be an appropriate combination for early osseointegration and implant use.

Transforming growth factor-beta1 incorporation in a calcium phosphate bone cement: material properties and release characteristics.

The bone regenerative properties of calcium phosphate cements (CPCs) may be improved by the addition of growth factors, such as recombinant human transforming growth factor-beta1 (rhTGF-beta1). Previously, we showed that rhTGF-beta1 in CPC stimulated the differentiation of preosteoblastic cells from adult rat long bones. The intermixing of rhTGF-beta1 in CPC, which was subsequently applied to rat calvarial defects, enhanced bone growth around the cement and increased the degradation of the cement. However, it is unknown whether the addition of rhTGF-beta1 changes the material properties of CPC and what the characteristics of the release of rhTGF-beta1 from CPC are. Therefore, we determined in this study the release of rhTGF-beta1, in vitro, from the cement pellets as implanted in the rat calvariae. The possible intervening effects of rhTGF-beta1 intermixing on the clinical compliance of CPC were studied through an assessment of its compressive strength and setting time, as well as its crystallinity, calcium-to-phosphorus ratio, porosity, and microscopic structure. We prepared CPC by mixing calcium phosphate powder (58% alpha-tricalcium phosphate, 25% anhydrous dicalcium phosphate, 8.5% calcium carbonate, and 8.5% hydroxyapatite) with a liquid (3 g/mL). The liquid for standard CPC consisted of water with 4% disodium hydrogen phosphate, whereas the liquid for modified CPC was mixed with an equal amount of 4 mM hydrochloride with 0.2% bovine serum albumin. The hydrochloride liquid contained rhTGF-beta1 in different concentrations for the release experiments. Most of the rhTGF-beta1 incorporated in the cement pellets was released within the first 48 h. For all concentrations of intermixed rhTGF-beta1 (100 ng to 2.5 mg/g of CPC), approximately 0.5% was released in the first 4 h, increasing to 1.0% after 48 h. Further release was only about 0.1% from 2 days to 8 weeks. CPC modification slightly increased the initial setting time at 20 degrees C from 2.6 to 5 min but had no effect on the final setting time of CPC at 20 degrees C or the initial and final setting times at 37 degrees C. The compressive strength was increased from 18 MPa in the standard CPC to 28 MPa in the modified CPC only 4 h after mixing. The compressive strength diminished in the modified CPC between 24 h and 8 weeks from 55 to 25 MPa. No other significant change was found with the CPC modification for rhTGF-beta1. X-ray diffraction revealed that standard and modified CPCs changed similarly from the original components, alpha-tricalcium phosphate and anhydrous dicalcium phosphate, into an apatite cement. The calcium-to-phosphorus ratio, as determined with an electron microprobe, did not differ for standard CPC and modified CPC. Standard and modified CPCs became dense and homogeneous structures after 24 h, but the modified CPC contained more crystal plaques than the standard CPC, as observed with scanning electron microscopy (SEM). SEM and back- scattered electron images revealed that after 8 weeks the cements showed equally and uniformly dense structures with microscopic pores (<1 microm). Both CPCs showed fewer crystal plaques at 8 weeks than at 24 h. This study shows that CPC is not severely changed by its modification for rhTGF-beta1. The prolonged setting time of modified cement may affect the clinical handling but is still within acceptable limits. The compressive strength for both standard and modified cements was within the range of thin trabecular bone; therefore, both CPCs can withstand equal mechanical loading. The faster diminishing compressive strength of modified cement from 24 h to 8 weeks likely results in early breakdown and so might be favorable for bone regeneration. Together with the beneficial effects on bone regeneration from the addition of rhTGF-beta1 to CPC, as shown in our previous studies, we conclude that the envisaged applications for CPC in bone defects are upgraded by the intermixing of rhTGF-beta1. Therefore, the combination of CPC and rhTGF-beta1 forms a promising synthetic bone graft.

Three-dimensional cartilage formation by bone marrow-derived cells seeded in polylactide/alginate amalgam.

Bone marrow-derived cells are considered as candidate cells for cartilage tissue engineering by virtue of their ability to undergo chondrogenesis in vitro when cultured in high density or when embedded within a three-dimensional matrix in the presence of growth factors. This study evaluated the potential of human bone marrow-derived cells for cartilage tissue engineering by examining their chondrogenic properties within a three-dimensional amalgam scaffold consisting of the biodegradable polymer, poly-L-lactic acid (PLA) alone, and with the polysaccharide gel, alginate. Cells were suspended either in alginate or medium and loaded into porous PLA blocks. Alginate was used to improve cell loading and retention within the construct, whereas the PLA polymeric scaffold provided appropriate mechanical support and stability to the composite culture. Cells seeded in the PLA/alginate amalgams and the plain PLA constructs were treated with different concentrations of recombinant human transforming growth factor-beta1 (TGF-beta 1) either continuously (10 ng/mL) or only for the initial 3 days of culture (50 ng/mL). Chondrogenesis was assessed at weekly intervals with cultures maintained for up to 3 weeks. Histological and immunohistochemical analysis of the TGF-beta 1-treated PLA/alginate amalgam and PLA constructs showed development of a cartilaginous phenotype from day 7 to day 21 as demonstrated by colocalization of Alcian blue staining with collagen type II and cartilage proteoglycan link protein. Expression of cartilage specific genes, including collagen types II and IX, and aggrecan, was detected in TGF-beta 1-treated cultures by reverse transcription-polymerase chain reaction analysis. The initiation and progression of chondrogenic differentiation within the polymeric macrostructure occurred with both continuous and the initial 3-day TGF-beta 1 treatment regimens, suggesting that key regulatory events of chondrogenesis take place during the early period of cell growth and proliferation. Scanning electron microscopy revealed abundant cells with a rounded morphology in the PLA/alginate amalgam. These findings suggest that the three-dimensional PLA/alginate amalgam is a potential candidate bioactive scaffold for cartilage tissue engineering applications.

Calcitonin responsiveness and receptor expression in porcine and murine osteoclasts: a comparative study

The presence of the calcitonin (CT) receptor is a distinguishing characteristic of osteoclasts; however, species variability exists with respect to functional responsiveness to CT. In the present study, CT responsiveness and temporal expression of the CT receptor in differentiating cultures of porcine osteoclasts was examined and compared to murine osteoclasts. In vitro porcine osteoclast differentiation was evaluated using bone marrow cultures from neonatal pigs. Murine osteoclast differentiation was studied using cocultures of murine bone marrow and BALC cells, a calvarial-derived cell line. In the presence of 1,25 (OH)2D3, a time-dependent increase in osteoclast differentiation was observed in porcine and murine cultures. Salmon CT (sCT) and porcine CT (pCT) inhibited 1,25 (OH)2D3-stimulated porcine osteoclast differentiation at 10−8 and 10−7 mol/L (60% with 10−7 mol/L sCT and 85% inhibition with 10−7 mol/L pCT). Treatment of murine cocultures with sCT (10−17–10−7 mol/L) resulted in a concentration-dependent decrease in osteoclast differentiation with a maximal inhibition of 70%. Osteoclast differentiation was inhibited in a concentration-dependent manner by recombinant human transforming growth factor-beta1 (rhTGF-β1) in both species. The effects of CT on resorption lacunae formation were determined by culturing in vitro generated porcine or murine osteoclasts on bovine cortical bone slices for 18 h in the presence or absence of CT. With both porcine and murine osteoclasts, a concentration-dependent decrease in resorption lacunae formation was observed between 10−13 and 10−7 mol/L sCT with the highest concentrations completely abolishing resorption. However, pCT only inhibited porcine osteoclastic resorption at 10−7 mol/L. CT receptor messenger ribonucleic acid (mRNA) expression was determined at different time points during in vitro osteoclast differentiation. In porcine cultures, expression of CT receptor mRNA correlated with the presence of osteoclasts. In murine cocultures, mRNA for the CT receptor was observed at each time point examined and was independent of the presence of multinucleated osteoclasts. Thus, porcine and murine differentiating osteoclast cultures express CT receptor mRNA; however, receptor expression correlates with osteoclast formation only in the porcine cultures. In summary, porcine and murine osteoclasts express CT receptor mRNA and functional responsiveness to CT. These findings suggest that the effects of sCT on osteoclast resorption are similar in murine and porcine cells, but that sCT is a less potent inhibitor of porcine than murine osteoclast differentiation.

Transforming growth factor-beta 1 enhances bone healing to unloaded tricalcium phosphate coated implants: an experimental study in dogs.

Growth of bone into cementless prosthetic components is compromised after revision of failed joint prostheses and by osteoporosis, gaps, and micromotion. We studied the effects of recombinant human transforming growth factor-beta 1 adsorbed on ceramic coated implants on the improvement of mechanical fixation and bone growth on the implant. Unloaded cylindrical grit-blasted titanium alloy implants were inserted bilaterally into both the medial and lateral femoral condyles of 10 skeletally mature mongrel dogs. The implants measured 10 mm in length and 6 mm in diameter and were initially surrounded by a 2 mm gap. One implant had an uncoated titanium surface and three implants were coated with tricalcium phosphate and 0, 0.3, or 3.0 micrograms of recombinant human transforming growth factor-beta 1. The dogs were killed at 6 weeks. Mechanical testing showed a 3-fold increase in fixation for the 0.3 microgram dose of recombinant human transforming growth factor-beta 1 and a 2-fold increase for the 3.0 micrograms dose. Histological analysis of bone growth on the implant demonstrated that maximal stimulation occurred with the 0.3 microgram dose, but bone volume in the gap was maximally stimulated by the 3.0 micrograms dose and increased 2-fold over control values. The majority of tricalcium phosphate was resorbed after the 6-week observation period. This study suggests that recombinant human transforming growth factor-beta 1 adsorbed onto implants coated with tricalcium phosphate ceramic can enhance mechanical fixation and bone growth on the implant. The use of transforming growth factor-beta 1 on ceramic coated prosthetic components may help to improve the functional outcome of cementless total joint replacements.

Influence of cytokine stimulation (granulocyte macrophage-colony stimulating factor, interleukin-3 and transforming growth factor-beta-1) on adhesion molecule expression in normal human bone marrow fibroblasts.

Interactions between stromal cells or extracellular matrix and hematopoietic cells are important factors for the very complex processes associated with differentiation and maturation in the bone marrow. To elucidate these multifold processes, the expression pattern of various adhesion molecules was studied on enriched fibroblastic populations derived from healthy volunteers. CD44 (homing cell adhesion molecule) and very late activation antigen beta 1 (VLA beta 1; CD29) could be demonstrated on almost all fibroblasts without an alteration following cytokine stimulation. On the other hand, VLA-2 (CDw49b), VLA-3 (CDw49c), VLA-4 (CDw49d), VLA-5 (CDw49e), intercellular adhesion molecule-1 (ICAM-1; CD54) and vascular cell adhesion molecule-1 (VCAM-1; CD106) were only represented by certain cell fractions. In our studies recombinant human granulocyte macrophage-colony stimulating factor failed to alter the expression pattern of these adhesion molecules, whereas recombinant human interleukin-3 (rhIL-3 showed a tendency for downregulation of the VLA antigens except for VLA beta 1. However, recombinant human transforming growth factor-beta 1 (rhTGF beta 1) exerted a reducing effect on the expression of VLA-3 and induced an increase in the VLA-5-positive fraction. In the immunoglobin class VCAM-1 revealed a decrease staining capacity after stimulation with rhTGF beta 1 and rhIL-3. Contrary to this finding, the presentation of ICAM-1 increased after administration of these mediators.

The proteoglycan synthesis repertoire of rabbit chondrocytes maintained in type II collagen gels

Repair of experimental articular cartilage lesions employing cultured rabbit articular chondrocytes requires a detailed knowledge of the phenotypic stability of these cells. A suitable matrix vehicle for use in chondrocyte transplantation is a much sought-after component of any transplantation paradigm. We studied the proteoglycan synthesis repertoire of young immature rabbit articular chondrocytes maintained in chick type II collagen gels or collagen gels supplemented with recombinant human transforming growth factor-beta 1 (rhTGF beta 1). Maintenance of chondrocytes in type II collagen gels increased the percentage 35SO4-labeled proteoglycans reaching equilibrium in the A1D1 or D1 fraction of CsCl density gradient when compared to chondrocytes maintained in polystyrene microwell cultures. Although rhTGF beta 1 supplementation increased the percentage of A1D1/D1 proteoglycan by chondrocytes grown on polystyrene, rhTGF beta 1 did not augment this percentage increase in A1D1/D1 when added to collagen II gels. Rabbit chondrocytes synthesized two core proteins derived from the high-density aggregatable proteoglycans. LI and LII have apparent molecular sizes of 480 kDa and 390 kDa, respectively. Both core protein forms were found in the medium fraction, but the predominant core protein form associated with the cell fraction was LI. Maintenance of chondrocytes in collagen II gels increased synthesis of both core proteins. In addition to the large core proteins, three other core proteins with properties on SDS PAGE characteristic of the small dermatan sulfate proteoglycans, biglycan and decorin, were identified. Synthesis of these core proteins was stimulated by maintenance in collagen gels. Furthermore, they were preferentially retained in the gel matrix. Chondrocytes maintained on glass or in type II collagen gels stained with monoclonal antibodies specific for chondroitin-6-sulfate, chondroitin-4-sulfate and keratan sulfate. However, while chondrocytes grown on glass slides failed to stain with monoclonal antibody 3B3 in the absence of chondroitinase ABC digestion, chondrocytes grown in collagen II gels stained intensely in the absence of enzyme pretreatment. These results were confirmed by Western blots.

One systemic administration of transforming growth factor-beta 1 reverses age- or glucocorticoid-impaired wound healing.

The role of intravenously administered recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) on the healing of incisional wounds in rats with impaired healing due to age or glucocorticoid administration was investigated. The administration of methylprednisolone to young adult rats decreased wound breaking strength to 50% of normal control. Breaking strength of incisional wounds from 19-mo-old rats was decreased approximately 27% compared with wounds from normal healing young adult rats. A single intravenous administration of rhTGF-beta 1 (100 or 500 micrograms/kg) increased wound breaking strength from old rats or young adult rats with glucocorticoid-induced impaired healing to levels similar to normal healing control animals when determined 7 d after injury. Even though the circulating half-life of systemically administered rhTGF-beta 1 is < 5 min, a sustained stimulatory effect on extracellular matrix secretion was evident in glucocorticoid-impaired rats when rhTGF-beta 1 was administered at the time of wounding, 4 h after wounding, or even 24 h before wounding. These observations indicate a previously unrecognized potential for the active form of TGF-beta 1 to profoundly influence the wound healing cascade after brief systemic exposure.

Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum.

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.

Development of a specific and sensitive two-site enzyme-linked immunosorbent assay for measurement of inhibin-A in serum

A polyclonal chicken antiserum against purified 32-kilodalton (kDa) recombinant inhibin-A (rh-InhA) and two monoclonal antibodies (mAb) against either rh-InhA (11B5) or 28-kDa recombinant activin-A (rh-ActA; 9A9) were used to develop three sensitive InhA enzyme-linked immunosorbent assays (ELISAs). The sensitivity of an ELISA using affinity-purified chicken anti-rh-InhA (Ck) for both coat and capture (Ck/Ck) averaged 78 +/- 3 pg/ml, while the mAb/Ck ELISAs (11B5/Ck or 9A9/Ck) averaged 100 +/- 6 pg/ml in a 10% serum matrix, with intra-and interassay coefficients of variation of 2-5% and 8-10%, respectively, for all assays. The ELISA formats did not cross-react with purified rh-ActA or recombinant human transforming growth factor-beta 1 or detect any immunoreactive proteins in medium conditioned by cell lines expressing rh-ActA or recombinant human transforming growth factor-beta 1. The Ck/Ck ELISA detected significant amounts of immunoreactivity in medium from cells expressing the free alpha-subunit of inhibin and recombinant inhibin-B (rh-InhB). In contrast, the mAb/Ck ELISAs showed no cross-reactivity to medium conditioned by these two cell lines. All three ELISA formats detected rh-InhA added to either human or rat serum in vitro or serum from rats injected with rhInhA. The Ck/Ck and 9A9/Ck ELISAs successfully quantitated inhibin in sera from patients undergoing ovulation induction and in rats (with or without sc administration of pregnant female serum gonadotropin). The 11B5/Ck ELISA appeared to be specific for the 32-kDa form of inhibin, while the 9A9/Ck ELISA was useful in quantitating inhibin-A in biological fluids, with little cross-reactivity to free alpha-chain or inhibin-B.