Plasma surface modification of poly(D,L-lactic acid) as a tool to enhance protein adsorption and the attachment of different cell types.

We have studied the influence of oxygen radio frequency glow discharge (RfGD) on the surface and bulk properties of poly(D,L-lactic acid) (PDLLA) and the effect of this surface modification on both protein adsorption and bone cell behavior. PDLLA films were characterized before and after plasma surface modification by water contact angle, surface energy, and adhesion tension of water as well as by scanning electron microscopy (SEM), X-ray electron spectroscopy (XPS), and Fourier transform infra-red (FTIR) spectroscopy. RfGD-films showed an increase in hydrophilicity and surface energy when compared with untreated films. Surface morphological changes were observed by SEM. Chemical analysis indicated significant differences in both atomic percentages and oxygen functional group. Protein adsorption was evaluated by combining solute depletion and spectroscopic techniques. Bovine serum albumin (BSA), fibronectin (FN), vitronectin (VN), and fetal bovine serum (FBS) were used in this study. RfGD-treated surfaces adsorbed more BSA and FN from single specie solutions than FBS that is a more complex, multi-specie solution. MG63 osteoblast-like cells and primary cultures of fetal rat calvarial (FRC) cells were used to assess both the effect of RfGD treatment and protein adsorption on cell attachment and proliferation. In the absence of preadsorbed proteins, cells could not distinguish between treated and untreated surfaces, with the exception of MG63 cells cultured for longer periods of time. In contrast, the adsorption of proteins increased the cells' preference for treated surfaces, thus indicating a crucial role for adsorbed proteins in mediating the response of osteogenic cells to the RfGD-treated PDLLA surface.

Modulating bone cells response onto starch-based biomaterials by surface plasma treatment and protein adsorption.

The effect of oxygen-based radio frequency glow discharge (rfGD) on the surface of different starch-based biomaterials (SBB) and the influence of proteins adsorption on modulating bone-cells behavior was studied. Bovine serum albumin, fibronectin and vitronectin were used in single and complex protein systems. RfGD-treated surfaces showed to increase in hydrophilicity and surface energy when compared to non-modified SBB. Biodegradable polymeric blends of cornstarch with cellulose acetate (SCA; 50/50wt%), ethylene vinyl alcohol (SEVA-C; 50/50wt%) and polycaprolactone (SPCL; 30/70wt%) were studied. SCA and SCA reinforced with 10% hydroxyapatite (HA) showed the highest degree of modification as result of the rfGD treatment. Protein and control solutions were used to incubate with the characterized SBB and, following this, MG63 osteoblast-like osteosarcoma cells were seeded over the surfaces. Cell adhesion and proliferation onto SCA was found to be enhanced for non-treated surfaces and on SCA+10%HA no alteration was brought up by the plasma modification. Onto SCA surfaces, BSA, FN and VN single solutions improved cell adhesion, and this same effect was found upscaled for ternary systems. In addition, plasma treated SEVA-C directed an increase in both adhesion and proliferation comparing to non-treated surfaces. Even though adhesion onto treated and untreated SPCL was quite similar, plasma modification clearly promoted MG63 cells proliferation. Regarding MG63 cells morphology it was shown that onto SEVA-C surfaces the variation of cell shape was primarily defined by the protein system, while onto SPCL it was mainly affected by the plasma treatment.

Augmentation of osseous phenotypes in vivo with a synthetic peptide.

The synthetic peptide B2A2-K-NS augmented the in vitro expression of osseous phenotypes when cells were stimulated with BMP-2, an osteoinductive growth factor. B2A2-K-NS significantly enhanced the effects of BMP-2-induced alkaline phosphatase activity and mineralization. In the absence of BMP-2, B2A2-K-NS did not have an effect on these endpoints. Based on these observations, in vivo studies were conducted to evaluate if B2A2-K-NS could augment osseous phenotypes in an osteoinductive environment in which BMP-2 should be present. In one study, human demineralized bone matrix (DBM) was used to generate an osteoinductive environment and the effects of B2A2-K-NS on ectopic mineralization of subcutaneous implants evaluated. In the second study, a noncritical sized defect in rabbit ulnas with inherent reparative capacity was used as the osteoinductive environment and was treated with or without B2A2-K-NS. In the DBM studies, B2A2-K-NS augmented mineralization as determined using a combination of radiographic analysis and von Kossa staining at 4 weeks postimplant. In the rabbit ulna model, B2A2-K-NS significantly increased the radiographic bone density in the defects compared to carrier-only or no-treatment controls after 6 weeks. Histological staining confirmed that B2A2-K-NS generated a pronounced bone repair response. The results are consistent with the hypothesis that B2A2-K-NS augments osseous phenotypes in an osteoinductive environment, and suggests that B2A2-K-NS may have clinical utility.

Effects of dissolved calcium and phosphorous on osteoblast responses.

The dissolution behavior of hydroxyapatite (HA) and its effect on the initial cellular response is of both fundamental and clinical importance. In this study, plasma-sprayed HA coatings were characterized by X-ray diffraction and Fourier transform infrared spectroscopy (FTIR). Calcium (Ca) and inorganic phosphorous (Pi) ions released from plasma-sprayed HA coatings within 3 weeks were measured by flame atomic absorption and colorimetrically molybdenum blue complex, respectively. To investigate the effect of dissolution of HA coatings on osteoblast response, additional Ca and Pi were added into the cell culture media to simulate the dissolution concentrations. Human embryonic palatal mesenchyme cells, an osteoblast precursor cell line, were used to evaluate the biological responses to enhanced Ca and Pi media over 2 weeks. Osteoblast differentiation and mineralization were measured by alkaline phosphatase-specific assay and 1,25 (OH)2 vitamin D3 stimulated osteocalcin production. The coatings exhibited an HA-type structure. FTIR indicated the possible presence of carbonates on the coatings. A dissolution study indicated a continual increase in Ca and Pi over time. In the cell culture study, enhanced osteoblast differentiation occurred in the presence of additional Ca concentration in the cell culture media. However, additional Pi concentration in the cell culture media was suggested to slow down osteoblast differentiation and mineralization.

Phospholipase A2 activating protein (PLAA) is required for 1alpha,25(OH)2D3 signaling in growth plate chondrocytes.

Phospholipase A2 (PLA2) is pivotal in the rapid membrane-mediated actions of 1,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3]. Microarray analysis indicated that PLA2 activating protein (PLAA) mRNA is upregulated 6-fold before rat growth plate cells exhibit 1alpha,25(OH)2D3-dependent protein kinase C (PKC) increases, suggesting that it plays an important role in 1alpha,25(OH)2D3's mechanism of action. PLAA mRNA was confirmed in 1alpha,25(OH)2D3-responsive growth zone (prehypertrophic and upper hypertrophic cell zones) chondrocytes by RT-PCR and Northern blot in vitro and by in situ hybridization in vivo. PLAA protein was shown by Western blot and immunohistochemistry. PLAAs role in 1alpha,25(OH)2D3 signaling was evaluated in growth zone cell cultures using PLAA peptide. Arachidonic acid release was increased as was PLA2-specific activity in plasma membranes and matrix vesicles. PKCalpha, but not PKCbeta, PKCepsilon, or PKCzeta, was increased. PLAAs effect was comparable to that of 1alpha,25(OH)2D3 and was additive with 1alpha,25(OH)2D3. PLA2 inhibitors quinacrine and AACOCF3, and cyclooxygenase inhibitor indomethacin blocked the effect of PLAA peptide on PKC, indicating arachidonic acid and its metabolites were involved. This was confirmed using exogenous arachidonic acid. Prostaglandin acted via EP1 based on inhibition by SC19220 and not via EP2 since AH6809 had no effect. Like 1alpha,25(OH)2D3, PLAA peptide also increased activity of phospholipase C-specific activity via beta-1 and beta-3 isoforms, but not delta-1 or gamma-1; the effect of PLAA was via lysophospholipid but not via arachidonic acid. PLAA peptide decreased [3H]-thymidine incorporation to 50% of the decrease caused by 1alpha,25(OH)2D3. In contrast, PLAA peptide increased alkaline phosphatase-specific activity and proteoglycan production in a manner similar to 1alpha,25(OH)2D3. This indicates that PLAA is a specific activator of PLA2 in growth plate chondrocytes, and suggests that it mediates the membrane effect of 1alpha,25(OH)2D3, thereby modulating physiological response.

Osteoblast precursor cell attachment on heat-treated calcium phosphate coatings.

The influence of properties of calcium phosphate (CaP) coatings on bone cell activity and bone-implant osseointegration is not well-established. This study investigated the effects of characterized CaP coatings of various heat treatments on osteoblast response. It was hypothesized that heat treatments of CaP coatings alter the initial osteoblast attachment. The 400 degrees C heat-treated coatings were observed to exhibit poor crystallinity and significantly greater phosphate or apatite species compared with as-sputtered and 600 degrees C heat-treated coatings. Similarly, human embryonic palatal mesenchyme (HEPM) cells, an osteoblast precursor cell line, seeded on 400 degrees C heat-treated coatings, exhibited significantly greater cell attachment compared with Ti surfaces, as-sputtered coatings, and 600 degrees C heat-treated coatings. The HEPM cells on Ti surfaces and heat-treated coatings were observed to attach through filopodia, and underwent cell division, whereas the cells on as-sputtered coatings displayed fewer filopodia extensions and cell damage. Analysis of the data suggested that heat treatment of CaP coatings affects cell attachment.

Experimental studies on bone induction using low-molecular-weight poly (DL-lactide-co-glycolide) as a carrier for recombinant human bone morphogenetic protein-2.

An appropriate carrier acting as a slow delivery vehicle for the BMPs is required for maximal clinical effectiveness of these bone-inductive proteins. The purpose of this study was to evaluate a low-molecular-weight PLGA copolymer as a synthetic, biodegradable carrier for rhBMP-2 implantation in vivo. Two, 10, or 50 microg of recombinant human BMP-2 were mixed with 10 mg of a poly (DL-lactide-co-glycolide) (PLGA) 50:50 copolymer and implanted into the calf muscles of Wistar rats. Soft X-ray analysis and histologic examination indicated that new bone formation occurred at all rhBMP-2-implanted sites within 3 weeks after implantation. Correlation of rhBMP-2 concentration with the amount of bone induction was confirmed by specific alkaline phosphatase activity and calcium content assay. In vitro analysis indicated that 78.5% of the PLGA copolymer was degraded to smaller molecular weight material after 14 days in PBS solution. It is suggested that rhBMP-2 was released in an active form at the implant site during the degradation of the copolymer, resulting in the induction of new bone formation. Thus this low-molecular-weight PLGA copolymer material represents a promising delivery vehicle for BMPs, and possibly other growth factors, around dental and orthopedic implants.

Bone response to radio frequency sputtered calcium phosphate implants and titanium implants in vivo.

The objective of this study was to evaluate the effect of radio frequency sputtered calcium phosphate (CaP) coatings of titanium (Ti) implants on the bond strength at the bone-implant interface and percent bone contact length. Cylindrical coated or noncoated implants (4.0-mm diameter by 8-mm long) were implanted for 3 and 12 weeks. At 3 weeks after implant placement, the ultimate interfacial strengths for as-deposited CaP-coated and heat-treated CaP-coated implants were 2.29 +/- 0.14 MPa and 1.28 +/- 0.04 MPa, respectively. These ultimate interfacial strength values at 3 weeks were statistically greater than the mean ultimate interfacial strength for control Ti implants (0.67 +/- 0.13 MPa). At 12 weeks after implant placement, no statistical differences in the mean ultimate interfacial strengths were observed between the as-deposited CaP-coated, heat-treated CaP-coated, and control Ti implants. Histomorphometric evaluation indicated greater percent bone contact lengths for the as-deposited CaP-coated implants compared with the heat-treated CaP-coated and control Ti implants 3 and 12 weeks after implant placement.

Cytotoxicity of intracanal bleaching agents on periodontal ligament cells in vitro.

Intracoronal bleaching of nonvital teeth is a simple and conservative procedure for esthetic restoration of discolored teeth. However it is possible that damage to the periodontal ligament may occur if the bleaching agents contact this tissue. The purpose of this study was to examine the cytotoxicity of intracanal bleaching agents on human periodontal ligament (PDL) cells in vitro. Three bleaching agents, 30% hydrogen peroxide (H2O2), 2.0 g/ml sodium perborate (SP) solution, and 2.0 g/ml SP in H2O2, were diluted from 10(-3) to 10(-7) with Eagle's minimal essential medium and incubated with PDL cells isolated and cultured from extracted teeth. Cytotoxicity was assessed quantitatively by determining the amount of lactic dehydrogenase activity released from the cells after exposure to the agents for 24 or 72 h. Dose-response curves were plotted, and TD50 values (dilution causing the release of 50% of control lactate dehydrogenase activity) and 95% confidence limits determined. The rank order of the TD50 values after exposure for 24 h was SP in H2O2 (most toxic) > H2O2 > SP solution (least toxic). After 72 h SP in H2O2 still produced the greatest cytotoxic effect. However the SP solution was more cytotoxic than H2O2 at this time point. It is concluded that the mixture of SP with H2O2 was the most toxic to the PDL cells in vitro.

PH changes at the surface of root dentin after intracanal dressing with calcium oxide and calcium hydroxide.

This study was designed to compare pH changes at the cervical and apical surfaces of root dentin after canal obturation with calcium oxide or calcium hydroxide pastes. The effect of the exposure to CO2 on the pH at the root surface also was assessed for both materials. Thirty-six extracted human canines were selected and instrumented. Wells 1 mm deep x 1.5 mm in diameter were drilled in the cervical and apical regions of the buccal root surface. The teeth were randomly divided into two groups and obturated with either calcium hydroxide or calcium oxide. pH was measured in the cervical and apical wells at varying time intervals until 48 days posttreatment. After pH measurement on day 48 the vials were flushed with CO2 and the pH measured again at days 53 and 68. The results indicated a similar pattern of pH increase after treatment with either calcium oxide or calcium hydroxide. pH dropped significantly after exposure to CO2 in both groups. This study demonstrated that hydroxyl ions produced when calcium oxide reacts with water diffuse through the dentinal tubules to the surface of the root in a manner similar to hydroxyl ions from calcium hydroxide.

Characterization of proliferation and cellular wound fill in periodontal cells using an in vitro wound model.

BACKGROUND: The therapeutic success of periodontal regenerative therapy may be compromised by our limited understanding of the wound healing process. Wound healing requires the coordination of complex cellular and molecular interactions. Recently, using an in vitro wound model, our laboratory has shown that gingival fibroblasts (GF) fill an in vitro wound more rapidly than periodontal ligament cells (PDL). This suggests that there may be differences in the levels of proliferation for these 2 cell types during the wound healing process. Such specific cell type differences may be significant in clinical outcomes of regenerative therapy. Therefore, the aim of this research was to characterize and compare the levels of both proliferation and cellular wound fill between GF and PDL using our in vitro wound model. METHODS: Primary cultures of human PDL and GF cells were established from explanted tissue, and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, or 9 days in media containing either 0.1% or 10% fetal bovine serum (FBS). At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU), fixed, and nuclei stained to measure DNA synthesis (as a measure for proliferation). Cells were counter stained with cytoplasmic stain to measure cell number. Quantitative analysis distant from (area of interest [AOI 1]), next to (AOI 2), and within the wound boundaries (AOI 3 and 4) was accomplished using computer-assisted histomorphometry. RESULTS: The levels of proliferation and cellular fill for each cell type were assessed relative to time and AOI. Overall, the PDL displayed greater (P <0.01) levels of proliferation than the GF. For both cell types, proliferation was found to be significantly (P<0.001) greater at day 2 compared to other time points. PDL displayed greater levels of proliferation than GF in all AOI, with this difference reaching significance (P<0.02) within the cell layer (AOI 1 and 2). When comparing levels of cellular fill in 10% FBS, GF displayed greater wound fill than the PDL. This difference was significant at day 6 (P <0.05) for both the marginal (AOI 3) and central (AOI 4) portions of the wound. CONCLUSIONS: These findings, demonstrating unique differences between PDL and GF with respect to proliferation and wound fill in an in vitro model, suggest that there may be cell-specific differences in cellular activity critical to periodontal wound healing. In addition, the results of this study show that the cellular proliferation response may not accurately reflect the overall wound healing response.

The effects of platelet-derived growth factor-BB on periodontal cells in an in vitro wound model.

BACKGROUND: Platelet-derived growth factor (PDGF-BB) has been shown to enhance periodontal regeneration. Principles of guided tissue regeneration dictate that one of the goals of therapy is to modulate the wound healing processes to favor repopulation of the wound with cells derived from the periodontal ligament rather than from the gingival tissues. Using an in vitro wound model, gingival fibroblasts (GF) have been shown to fill a wound space significantly faster than periodontal ligament cells (PDL). There are no data reported directly comparing the response of these 2 cell types to PDGF-BB within such a wound model. Therefore, the aims of this research were: 1) to characterize both the proliferative and wound fill (WF) effects of PDGF-BB within an in vitro model and 2) to compare specific growth factor effects between GF and PDL. METHODS: Primary cultures of both human PDL and GF were derived from explanted tissues and passaged to 12-well tissue culture plates. Triplicate cultures of both cell types were grown to confluence and in vitro wounds were mechanically created, removing a 3 mm wide band of the cell layer across the diameter of the wells. The wells were then incubated for 2, 6, and 9 days in media containing 0.1% fetal bovine serum (FBS) and 1 of 5 concentrations of PDGF-BB. At each time point, cells were pulsed with 5-bromo, 2-deoxyuridine (BrdU) fixed, and nuclei were stained to measure BrdU incorporation (as a measure for proliferation). Cells were counter-stained with cytoplasmic stain to measure cell number. Quantitative analyses within the wound boundaries, marginally (area of interest [AOI] 1) and centrally (AOI 2), were accomplished using computer-assisted histomorphometry. RESULTS: PDL exhibited a significantly greater proliferative response to PDGF-BB in both AOI when compared to GF (P <0.0001). The PDL exhibited increased levels of proliferation at concentrations of PDGF-BB greater than or equal to 10 ng/ml. By contrast, GF displayed no increase in proliferation in response to stimulation with PDGF-BB at any of the concentrations tested when compared to negative controls. The wound fill (WF) responses to PDGF-BB were similar between PDL and GF, with both cell types responding in an all or none fashion when measured at day 2, and in a concentration-dependent manner at later time points. The only significant difference in WF between PDL and GF occurred in AOI 2 in negative control medium (0 ng/ml of PDGF-BB), with GFs having greater (P <0.01) levels of WF over the 9 days. CONCLUSION: The findings from this study demonstrate differing effects of PDGF-BB on the proliferation of PDL and GF in this in vitro model. These results suggest that there may be cell-specific differences critical to periodontal wound healing that may be exploited in the development of new therapies.

Tissue response and osteoinduction of human bone grafts in vivo.

Freeze-dried human bone allograft is used clinically as an adjunct to autologous bone graft. When freeze-dried human bone allograft is demineralized, the allograft is osteoinductive, since it causes bone to form heterotopically. Both types of allograft are also used alone, such as in spinal fusions, critical size defects, and periodontal therapy. The purpose of this study was to determine the effect of demineralization on the osteoinductive potential of human bone grafts obtained from two different groups of patients. One group consisted of six patients younger than 42 years of age, while the other group consisted of six patients who were older than 70 years of age. The harvested material was lyophilized and divided into two portions, one of which was used directly while the other was demineralized. Osteoinductive ability was established using an in vivo assay for heterotopic bone formation. Activity in these bone grafts was compared with a batch of commercially prepared demineralized, freeze-dried human bone grafts that had been previously shown to be active and another batch that had been shown to display low ('inactive') osteoinductive ability. A bone induction score was determined for each group of grafts based on the number and size of any ossicles formed. In addition, the area of new bone formation and area of residual particles were determined histomorphometrically. Tissue response to the bone grafts varied with donor age and whether the samples had been demineralized or not. Only demineralized, freeze-dried bone graft from patients younger than 42 years of age was osteoinductive; all other batches displayed little or no osteoinductive activity. In the demineralized, freeze-dried bone from donors younger than 42 years of age, the bone induction score and new bone area were significantly higher than in the other batches of bone graft, and the area of residual particles was reduced. Both demineralized and nondemineralized bone graft from patients older than 70 years of age were encapsulated in dense, fibrous connective tissue. These results may help explain the observed differences in clinical outcome when demineralized, freeze-dried bone graft or nondemineralized, freeze-dried bone graft from different donors is used in bone regeneration applications.

Ability of deproteinized cancellous bovine bone to induce new bone formation.

BACKGROUND: Preclinical and clinical studies indicate that deproteinized cancellous bovine bone is osteoconductive and may be osteopromotive. Previous studies using commercial preparations failed to demonstrate the presence of protein, implicating bone-mineral composition and 3-dimensional structure as reasons for clinical success; however, these studies did not examine whether osteoinductive factors might be present in close association with the mineral phase. METHODS: Deproteinized cancellous bovine bone was decalcified and any protein present released by chaotropic solvents using the protocol described for purification of bone morphogenetic proteins (BMPs). Three extracts were obtained and tested for their ability to support osteoinduction in the calf muscle of nude mice. RESULTS: Protein content averaged 11 microg/g based on absorbance at 280 nm using bovine serum albumin as a standard. All extracts contained material that stained positively with silver stain after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Western blots of these gels indicated the presence of transforming growth factor-beta (TGF-beta) and BMP-2. All 3 extracts were osteoinductive in the nude mouse model when combined with inactive DFDBA, and bone formation was comparable to that induced by active DFDBA. Deproteinized cancellous bovine bone by itself was not osteoinductive in the nude mouse, but in a clinical case, exhibited osteoclastic resorption with adjacent new bone formation. CONCLUSIONS: The results suggest that small amounts of protein are present in deproteinized cancellous bovine bone in close association with the mineral phase. Some of the extracted material has osteoinductive potential and may contain growth factors. This may explain the osteopromotive ability of deproteinized cancellous bovine bone clinically.

Porcine fetal enamel matrix derivative enhances bone formation induced by demineralized freeze dried bone allograft in vivo.

BACKGROUND: Embryonic enamel matrix proteins are involved in the formation of acellular cementum during development of the periodontal attachment apparatus, suggesting that these proteins might be used clinically to promote periodontal regeneration. At present, it is unknown if these proteins are osteoinductive, osteoconductive, or osteopromotive. To address this question, we examined the ability of a commercially prepared embryonic porcine enamel matrix derivative to induce new bone formation in nude mouse calf muscle, or to enhance the bone induction ability of a demineralized freeze-dried bone allograft (DFDBA). METHODS: Porcine fetal enamel matrix derivative (EMD) was implanted bilaterally in the calf muscle of 4 male Nu/Nu mice per treatment group (N = 8 implants): 2 mg EMD alone; 4 mg EMD alone; inactive human DFDBA alone; inactive DFDBA + 2 mg EMD; inactive DFDBA + 4 mg EMD; active DFDBA alone; active DFDBA + 2 mg EMD; and active DFDBA + 4 mg EMD. Implants were harvested after 56 days and examined histologically for bone induction using a semi-quantitative score and histomorphometrically for area of new bone, cortical bone, bone marrow, and residual DFDBA. RESULTS: Implants containing inactive DFDBA, 2 mg EMD, 4 mg EMD, and inactive DFDBA + 2 or 4 mg EMD did not induce new bone. Active DFDBA and active DFDBA + 2 mg EMD induced new bone to a similar extent. In contrast, active DFDBA + 4 mg EMD resulted in enhanced bone induction, area of new bone, and cortical bone. Residual DFDBA was also increased in this group. CONCLUSIONS: EMD is not osteoinductive. However, it is osteopromotive, due in part to its osteoconductive properties, but a threshold concentration is required.

Porcine fetal enamel matrix derivative stimulates proliferation but not differentiation of pre-osteoblastic 2T9 cells, inhibits proliferation and stimulates differentiation of osteoblast-like MG63 cells, and increases proliferation and differentiation of normal human osteoblast NHOst cells.

BACKGROUND: Embryonic enamel matrix proteins are hypothesized to be involved in the formation of acellular cementum during tooth development, suggesting that these proteins can be used to regenerate periodontal tissues. Enamel matrix protein derived from embryonic porcine tooth germs is used clinically, but the mechanisms by which it promotes the formation of cementum, periodontal ligament, and bone are not well understood. METHODS: This study examined the response of osteoblasts at 3 stages of osteogenic maturation to porcine fetal enamel matrix derivative (EMD). Proliferation (cell number and [3H]-thymidine incorporation), differentiation (alkaline phosphatase and osteocalcin), matrix synthesis ([35S]-sulfate incorporation; percentage of collagen production), and local factor production (prostaglandin E2 [PGE2] and transforming growth factor-beta 1 [TGF-beta1]) were measured in cultures of 2T9 cells (pre-osteoblasts which exhibit osteogenesis in response to bone morphogenetic protein-2 [BMP-2]), MG63 human osteoblast-like osteosarcoma cells, and normal human osteoblasts (NHOst cells). RESULTS: EMD regulated osteoblast proliferation and differentiation, but the effects were cell-specific. In 2T9 cell cultures, EMD increased proliferation but had no effect on alkaline phosphatase-specific activity. EMD decreased proliferation of MG63 cells and increased cellular alkaline phosphatase and osteocalcin production. There was no effect on collagen synthesis, proteoglycan sulfation, or PGE2 production; however, TGF-beta1 content of the conditioned media was increased. There was a 60-fold increase in cell number in third passage NHOst cells cultured for 35 days in the presence of EMD. EMD also caused a biphasic increase in alkaline phosphatase that was maximal at day 14. CONCLUSIONS: EMD affects early states of osteoblastic maturation by stimulating proliferation, but as cells mature in the lineage, EMD enhances differentiation.

Pretreatment with platelet derived growth factor-BB modulates the ability of costochondral resting zone chondrocytes incorporated into PLA/PGA scaffolds to form new cartilage in vivo.

Optimal repair of chondral defects is likely to require both a suitable population of chondrogenic cells and a biodegradable matrix to provide a space-filling structural support during the early stages of cartilage formation. This study examined the ability of chondrocytes to support cartilage formation when incorporated into biodegradable scaffolds constructed from copolymers (PLG) of polylactic acid (PLA) and polyglycolic acid (PGA) and implanted in the calf muscle of nude mice. Scaffolds were fabricated to be more hydrophilic (PLG-H) or were reinforced with 10% PGA fibers (PLG-FR), increasing the stiffness of the implant by 20-fold. Confluent primary cultures of rat costochondral resting zone chondrocytes (RC) were loaded into PLG-H foams and implanted intramuscularly. To determine if growth factor pretreatment could modulate the ability of the cells to form new cartilage, RC cells were pretreated with recombinant human platelet derived growth factor-BB IPDGF-BB) for 4 or 24 h prior to implantation. To assess whether scaffold material properties could affect the ability of chondrogenic cells to form cartilage, RC cells were also loaded into PLG-FR scaffolds. To determine if the scaffolds or treatment with PDGF-BB affected the rate of chondrogenesis, tissue at the implant site was harvested at four and eight weeks post-operatively, fixed, decalcified and embedded in paraffin. Sections were obtained along the transverse plane of the lower leg, stained with haematoxylin and eosin, and then assessed by morphometric analysis for area of cartilage, area of residual implant, and area of fibrous connective tissue formation (fibrosis). Whether or not the cartilage contained hypertrophic cells was also assessed. The amount of residual implant did not change with time in any of the implanted tissues. The area occupied by PLG-FR implants was greater than that occupied by PLG-H implants at both time points. All implants were surrounded by fibrous connective tissue, whether they were seeded with RC cells or not. The amount of fibrosis was reduced at eight weeks for both implant types. When RC cells were present, the amount of fibrosis was less than seen in cell-free scaffolds. Pretreatment with PDGF-BB caused a slightly greater degree of fibrosis at four weeks than was seen if untreated cells were used in the implants. However, at eight weeks, if the cells had been exposed to PDGF-BB for 24 h, fibrosis was comparable to that seen associated with cell-free scaffolds. The cells supported an equivalent area of cartilage formation in both scaffolds. PDGF-BB caused a time-dependent decrease in cartilage formation at four weeks, but at eight weeks, there was a marked increase in cartilage formation in PDGF-BB-treated cells that was greatest in cells exposed for 4 h compared to those exposed for 24 h. Moreover, PDGF-BB decreased the formation of hypertrophic cells. The results indicate that in this model, RC cells produce cartilage; pretreatment of the RC cells with PDGF-BB promotes retention of a hyaline-like chondrogenic phenotype; and the material properties of the implant do not negatively impact on the ability of the cells to support chondrogenesis.

Time course of the increase in trk A expression in trigeminal neurons after tooth injury.

Injury to tooth pulp often results in extensive sprouting of sensory nerve fibers at the site of wound repair due to local increases in nerve growth factor (NGF) concentration. NGF interacts with high-affinity binding sites, termed trk A receptors, located on the cell membranes of responsive neurons. If NGF induces wound repair and/or nociceptive responses in tooth pulp, then changes in expression of NGF receptors (trk A receptors) in response to dentin injury would be expected. To characterize the role of trk A receptors in mediating NGF-induced signals to sensory neurons, trigeminal ganglia from adult male rats were examined for changes in expression of trk A as a function of time after injury to maxillary molar dentin. In situ hybridization was performed with 35S-labeled riboprobes encoding the sense or antisense trk A sequences, and grain densities quantified over maxillary neurons. As early as 12 h after tooth injury, grain density counts increased by 71% above control level, indicating an increase in trk A receptor mRNA expression. Grain densities obtained from ganglia harvested at all time points through 168 h after injury remained elevated. At 336 h (14 days) after injury, trk A receptor expression had decreased such that grain density counts were not different from preinjury levels. Thus our results suggest that NGF may be mediating repair and pain responses by the sustained upregulation of its cell surface receptor, trk A, in neurons of the trigeminal ganglia.

BMP stimulation of bone response adjacent to titanium implants in vivo.

The objective of this study was to evaluate the effect of bone morphogenetic protein (BMP) on the bond strength of titanium (Ti) implants at the bone-implant interface. Thirty-six Brånemark screw implants (3.75 mm diameter by 15 mm long) were implanted for 3 and 12 weeks. At 3 weeks after implantation, the mean reverse torque value for implants stimulated with BMP delivered using an atelopeptide type-I collagen carrier (74.2 +/- 5.2 N cm) was observed to be statistically greater (P < 0.000016) than the mean reverse torque value for control Ti implants (32.8 +/- 1.1 N cm). Similarly, at 12 weeks after implantation, the mean reverse torque value for implants stimulated with BMP (89.2 +/- 2.7 N cm) was statistically greater (P < 0.0042) than the mean reverse torque value for control Ti implants (75.8 +/- 2.4 N cm). In addition, histomorphometric evaluations indicated more bone contact with the BMP stimulated implant surfaces compared to the controls after 3 weeks implantation. It was concluded from this study that the use of BMP-atelopeptide type-I collagen mixture is an effective means of obtaining greater bond strength at the bone implant interface within a shorter time period than Ti implants without BMP.

Potential of porous poly-D,L-lactide-co-glycolide particles as a carrier for recombinant human bone morphogenetic protein-2 during osteoinduction in vivo.

Several different biodegradable bone graft materials are in clinical or preclinical use for the repair of bone defects in orthopedics, maxillofacial surgery, and periodontics. This study tested the hypothesis that poly-D,L-lactide-co-glycolide copolymer (PLG) can be used as an effective carrier of recombinant human bone morphogenetic protein-2 (rhBMP-2) and that the composite has osteoinductive ability. Porous PLG rods were shredded to a particle size ranging from 250 to 850 microm. Active and inactive demineralized freeze-dried bone allografts (DFDBA) with a comparable particle size were used as positive and negative controls, respectively. PLG particles were treated with vehicle or with 5 or 20 microg rhBMP-2. DFDBA and PLG particles were placed in gelatin capsules, mixed with vehicle or rhBMP-2, and implanted at intramuscular sites in male Nu/Nu (nude) mice. Each mouse underwent bilateral implantation with implants of the same formulation, resulting in five groups of four mice per group: active DFDBA, inactive DFDBA, PLG, PLG + 5 microg rhBMP-2, and PLG + 20 microg rhBMP-2. After 56 days, the implants were recovered and processed for histology. Bone induction was assessed by use of a semiquantitative scoring system based on the amount of new bone formed in representative histological sections. Histomorphometry was also used to measure the area of new bone formed and the area of residual implant material. The results showed that active DFDBA induced the formation of ossicles containing new bone with bone marrowlike tissue, whereas inactive DFDBA or PLG particles alone did not induce new bone. The addition of rhBMP-2 to PLG particles resulted in new bone formation that had a greater bone induction score than active DFDBA. Moreover, the histomorphometric analysis showed that the addition of rhBMP-2 to PLG particles induced the formation of a greater area of new bone and bone marrowlike tissue than active DFDBA. The resorption of the PLG particles was markedly increased with the addition of rhBMP-2, suggesting that rhBMP-2 may attract and regulate resorptive cells at the implantation site. The results of the present study indicate that PLG copolymers are good carriers for BMP and promote the induction of new bone formation. Further, the PLG copolymers with rhBMP-2 had a greater effect in inducing new bone formation and resorbing the implanted material than active DFDBA alone.