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PubMed2003

Extracellular matrix production by human osteoblasts cultured on biodegradable polymers applicable for tissue engineering.

The nature of the extracellular matrix (ECM) is crucial in regulating cell functions via cell-matrix interactions, cytoskeletal organization, and integrin-mediated signaling. In bone, the ECM is composed of proteins such as collagen (CO), fibronectin (FN), laminin (LM), vitronectin (VN), osteopontin (OP) and osteonectin (ON). For bone tissue engineering, the ECM should also be considered in terms of its function in mediating cell adhesion to biomaterials. This study examined ECM production, cytoskeletal organization, and adhesion of primary human osteoblastic cells on biodegradable matrices applicable for tissue engineering, namely polylactic-co-glycolic acid 50:50 (PLAGA) and polylactic acid (PLA). We hypothesized that the osteocompatible, biodegradable polymer surfaces promote the production of bone-specific ECM proteins in a manner dependent on polymer composition. We first examined whether the PLAGA and PLA matrices could support human osteoblastic cell growth by measuring cell adhesion at 3, 6 and 12h post-plating. Adhesion on PLAGA was consistently higher than on PLA throughout the duration of the experiment, and comparable to tissue culture polystyrene (TCPS). ECM components, including CO, FN, LM, ON, OP and VN, produced on the surface of the polymers were quantified by ELISA and localized by immunofluorescence staining. All of these proteins were present at significantly higher levels on PLAGA compared to PLA or TCPS surfaces. On PLAGA, OP and ON were the most abundant ECM components, followed by CO, FN, VN and LN. Immunofluorescence revealed an extracellular distribution for CO and FN, whereas OP and ON were found both intracellularly as well as extracellularly on the polymer. In addition, the actin cytoskeletal network was more extensive in osteoblasts cultured on PLAGA than on PLA or TCPS. In summary, we found that osteoblasts plated on PLAGA adhered better to the substrate, produced higher levels of ECM molecules, and showed greater cytoskeletal organization than on PLA and TCPS. We propose that this difference in ECM composition is functionally related to the enhanced cell adhesion observed on PLAGA. There is initial evidence that specific composition of the PLAGA polymer favors the ECM. Future studies will seek to optimize ECM production on these matrices for bone tissue engineering applications.

Biodegradation, Environmental↗

PubMed2003

Osteoblastic cell response to thin film of poorly crystalline calcium phosphate apatite formed at low temperatures.

The response of osteoblastic cells to a thin film of poorly crystalline calcium phosphate apatite crystals (PCA) was examined in vitro. The PCA thin film was prepared on polystyrene culture dishes using highly metastable calcium phosphate ion solution at low temperatures. The PCA thin film was formed through fusion and transformation of granular calcium phosphate particles, which had initially formed on the surface, into a film of calcium phosphate apatite crystal. The PCA thin film was used for cell culture without additional surface treatment. The osteoblastic cell behaviors including adhesion, proliferation, expression of the marker genes, and calcified matrix formation were examined on the PCA thin film using primary osteoblasts or MC3T3-E1 cells. The cells were well attached and had spread in a slender shape over the PCA thin film. The extent of cell proliferation on the PCA thin film is as much as on the plain dishes. In addition, a much larger number of calcified nodules had formed on the PCA thin film than on the plain dish. The expression of the marker genes such as alkaline phosphatase, osteocalcin, osteopontin, osteonectin was apparent. These results demonstrate that the osteoblasts exhibit a full spectrum of cellular activity including the adequate differentiation on the PCA thin film. Therefore, a PCA thin film can be used as a coating material for biomaterials where the surface is not adequate for inducing the full activity of bone cells.

3T3 Cells↗

PubMed2004

The functional expression of human bone-derived cells grown on rapidly resorbable calcium phosphate ceramics.

The use of biodegradable bone substitutes is advantageous for alveolar ridge augmentation, since it avoids second-site surgery for autograft harvesting. This study examines the effect of novel, rapidly resorbable calcium phosphates on the expression of bone-related genes and proteins by human bone-derived cells (HBDC) and compares this behavior to that of tricalciumphosphate (TCP). Test materials were alpha-TCP, and four materials which were created from beta-Rhenanite and its derivatives: R1-beta-Rhenanite (CaNaPO(4)); R1/M2 composed of CaNaPO(4) and MgNaPO(4); R1+SiO(2) composed of CaNaPO(4) and 9% SiO(2) (wt%); and R17-Ca(2)KNa(PO(4))(2). HBDC were grown on the substrata for 3, 5, 7, 14 and 21 days, counted and probed for various mRNAs and proteins (Type I collagen, osteocalcin, osteopontin, osteonectin, alkaline phosphatase and bone sialoprotein). All substrata supported continuous cellular growth for 21 days. At day 21, surfaces of R1+SiO(2) and R17 had the highest number of HBDC. At 14 and 21 days, cells on R1 and on R1+SiO(2) displayed significantly enhanced expression of all osteogenic proteins. Since all novel calcium phosphates supported cellular proliferation together with expression of bone-related proteins at least as much as TCP, these ceramics can be regarded as potential bone substitutes. R1 and R1+SiO(2) had the most effect on osteoblastic differentiation, thus suggesting that these materials may possess a higher potency to enhance osteogenesis than TCP.

Bone Substitutes↗

PubMed2000

A long-term study of implanted artificial hydroxyapatite particles surrounding the carotid artery in adult dogs.

In this long-term study, we implanted HAP into adult dogs using a silicone chamber attached to the carotid artery to clarify tissue reaction to HAP implantation over a long period. We designed chambers and both hemispheres of the chambers were filled with HAP particles, and were placed around both carotid arteries of seven adult dogs. The implants were removed after 150, 300, 380 days, and histological and ultrastructual examination was undertaken. We observed bone-like tissue which was formed where HAP particles were implanted. Immunohistochemical findings showed that osteocalcin and osteonectin were as positive in the bone-like tissue as in normal bone. This study suggests that biological factors from the arterial wall might play an important role in new bone-like tissue forming, and that HAP has a strong osteoconductive ability even at heterogeneous sites.

Animals↗

PubMed2001

Matrix metalloproteinase-1, -3, -13 and aggrecanase-1 and -2 are differentially expressed in experimental osteoarthritis.

The aim of this study was to characterize the cellular phenotypes of articular cartilage and meniscus in rabbits with experimentally induced osteoarthritis (OA), by histological and molecular biological techniques. OA was induced by severing the anterior cruciate ligament of the knee and rabbits were killed 2, 4 or 9 weeks following surgery. Our histological observations show a progressive destruction of extracellular matrix in both tissues. To determine whether these morphological changes could be related to alterations in the regulation of gene expression for a subset of relevant molecules, levels of mRNA for proteinases and one inhibitor (MMP-1, -3 and -13, aggrecanase-1 and -2 and TIMP-1), matrix molecules and one chaperone (type II and X collagens, aggrecan, osteonectin, betaig-h3 and BiP) were assessed by reverse transcription-polymerase chain reaction. Our results indicate that for most markers expression profiles were similar in both tissues. In particular, matrix protein gene expression remained stable or varied little during progression of OA, suggesting a poor repair capacity of the tissues. MMP gene expression increased rapidly whereas aggrecanase gene expression remained stable. These findings suggest that differential regulation of mRNA levels of MMP-1, -3 and -13 on the one hand and aggrecanase-1 and -2 on the other, occurs during OA.

ADAM Proteins↗

PubMed1997

Neutral lipids facilitate transfer of bone morphogenetic proteins and other noncollagenous proteins.

In the process of separation of bone morphogenetic proteins from bone matrix, lipids were found in unexpected amounts closely associated with noncollagenous proteins soluble in guanidine hydrochloride. Lipids representing 33.7-49.9% by weight were recovered with various solvents. Composites of noncollagenous proteins and lipids soluble in either chloroform- methanol or acetone implanted in the hindquarter muscles of mice induced the formation of large deposits of heterotopic bone. The protein-lipid aggregates formed microspherules which were stained by Sudan Black B. Implants of bone morphogenetic proteins and noncollagenous proteins-lipid microspherules stained with Sudan Black B induced bone development in the same manner as unstained delipidized bone morphogenetic proteins associated with noncollagenous proteins. Lipid-free osteocalcein, osteonectin, albumin and other bone matrix proteins did not induce bone formation or bind Sudan Black B. The more highly purified the noncollagenous proteins, with or without activity of bone morphogenetic proteins, the lower the level of binding with Sudan Black B. Acetone-soluble bone matrix lipids consisted chiefly of triglycerides, cholesterol and saturated short chain fatty acids, and included little or no phospholipids or monounsaturated fatty acids. Composites of recombinant bone morphogenetic proteins-2 and acetone-soluble lipids induced larger deposits of bone than implants of recombinant bone morphogenetic proteins-2 without acetone-soluble lipids. The hypothesis that an association of bone lipids with protein facilitates the local transport of bone morphogenetic proteins warrants further investigation.

Animals↗

PubMed1995

New aspects of cell biology in osteosarcoma.

Among the solid tumors of childhood and adolescence, osteosarcoma (OS) represents the most prominent example of efficient aggressive chemotherapy with secondary surgical therapy. A specific subclassification of the tumor is indispensable and must include recent results of cell biology. The co-distribution of different collagen types I-VI reflects the diverse differentiation of osteosarcoma cells, supporting the concept of a pluripotent mesenchymal cell to be the stem cell of the tumor. In contrast, osteonectin (SPARC) may not be considered as a reliable marker for osteosarcoma. The experience of special proteins being secreted by osteosarcoma cells is rather limited. Detailed molecular biological studies are still lacking. A loss of alleles on chromosome 17, particularly in the defined region 17p 13, can be observed in more than 75% of all OS, suggesting the contribution of a tumor suppressor gene, p53, located in that region. It is a 53 kd nucleophosphoprotein binding the major transforming protein, the large T antigen of Simian Virus 40. Immunohistological results showed positive staining with the antibody Pab 240 in 13 of 18 cases. In one osteoblastic OS, a novel splice mutation resulting in a fusing of exon 5 directly to exon 7 was detected. RB1 gene is also of major importance for the tumorigenesis of OS. The multidrug resistance (mdr) is associated with a membrane-bound channel-forming transport protein, the P-glycoprotein. It is a conserved plasma membrane component of about 170 kd. Both the human isoforms mdr 1 and mdr 3 are localised in the long arm of chromosome 7.(ABSTRACT TRUNCATED AT 250 WORDS)

Bone Neoplasms↗

PubMed1997

Osteopontin is associated with bioprosthetic heart valve calcification in humans.

Calcification of non-osseous tissues such as heart valves or vessels is a major concern in clinical practice. The exact mechanism is still unknown. Numerous studies have shown that mineral deposits of crystalline hydroxyapatite within these tissues were associated with increased non-collagenous protein content. More recently osteopontin was found to be associated with calcification in living tissues such as vessels and native human aortic valves. The aim of this study was to determine whether or not non-collagenous proteins can also be found in non-living tissues such as glutaraldehyde-pretreated porcine valves after implantation in humans. Thirty-eight glutaraldehyde pretreated porcine bioprostheses were studied: 16 not implanted and 22 after 11 years of implantation in the aortic and mitral valve position in humans. In areas of calcification vizualized by Von Kossa staining and microradiography, immunostaining using polyclonal antibodies against calcium-binding proteins showed osteopontin positive staining and no staining for osteocalcin, bone sialoprotein or osteonectin. In uncalcified areas and in non-implanted values, staining for osteopontin or other calcium-binding proteins was negative. Western blot analysis of macroscopically calcified and uncalcified areas showed that several proteins were adsorbed in implanted values and confirmed the presence of osteopontin in the calcified areas, while no immunolabelling was found in non-calcified areas, in uncalcified valves and in non-implanted valves. Thus the presence of osteopontin in the calcified areas of bioprosthetic heart valves implanted in human indicates that this protein is associated with bioprosthetic valvular calcification. Since these values are made of non-living connective tissue, and no cell immunostained for osteopontin was found around the calcified area, this suggests that a non-cellular mediated mechanism involving protein adsorption may play a role in bioprosthetic valvular calcification.

Bioprosthesis↗

PubMed1991

Biosynthesis of bone proteins by fetal porcine calvariae in vitro. Rapid association of sulfated sialoproteins (secreted phosphoprotein-1 and bone sialoprotein) and chondroitin sulfate proteoglycan (CS-PGIII) with bone mineral.

To study the biosynthesis of bone proteins, fragments of fetal porcine calvariae were cultured in the presence of 50 micrograms/ml ascorbate and 10 mM beta-glycerophosphate and individual cultures labeled for either 4 h or 48 h with [35S]-methionine, Na2[35SO4], Na3[32PO4] or [14C]-glycine plus [14C]-proline. The radiolabeled proteins in tissue extracts were obtained by sequential extraction with 4 M GuHCl (G1-extract), 0.5 M EDTA (E-extract), and again with 4 M GuHCl (G2-extract) and analyzed together with the radiolabeled proteins secreted into the medium. SPP-1 (secreted phosphoprotein 1, osteopontin) was the major non-collagenous protein deposited into the bone matrix, with lesser amounts of BSP (bone sialoprotein), osteocalcin and chondroitin sulfate proteoglycans (CS-PG II and CS-PG III). SPP-1 was also the major phosphorylated protein and was recovered, together with several fragmented forms, almost entirely in the demineralizing extracts. Moreover, approximately one-half of the [35SO4] incorporated into E-extract proteins was present in SPP-1, the remainder being incorporated into PGs with smaller amounts associated with BSP. Over 65% of the [35SO4] in the proteoglycans of the demineralizing extracts was recovered in the small CS-PG III with less than 35% in CS-PG II, the bone homologue of DS-PG II (decorin). In contrast, CS-PG II was the predominant small proteoglycan in culture media and in guanidine extracts. Some sulfated BSP was also observed in guanidine extracts and small amounts appeared to bind to collagen. Radiolabeled SPARC (osteonectin), a prominent protein of fetal porcine bone, was not detected in the mineralized bone tissues but was prominent in the culture medium. These results demonstrate that following secretion, the major proteins expressed by osteoblastic cells are initially incorporated into different tissue compartments, with most of the sulfated sialoproteins and CS-PG III associating rapidly with the hydroxyapatite crystals. The initial distribution of these proteins is of importance in the evaluation of their role in bone formation and mineralization.

Animals↗

PubMed2001

MC3T3-E1-conditioned medium-induced mineralization by clonal rat dental pulp cells.

Dental pulp is thought to participate in supplementary mineralization, such as reparative dentin and pulp stones, but no direct proof of this has been reported. To study this process at a molecular level, we investigated the matrix mineralization of dental pulp using a clonal cell line (RPC-C2A) derived from rat incisor dental pulp. Mineralized nodules in extracellular matrix were formed by RPC-C2A cells cultured in the presence of conditioned medium (CM) from confluent osteoblastic MC3T3-E1 cells. These nodules were stained by the von Kossa method and with alizarin red S and quantified by the measurement of acid-soluble calcium deposition. This CM was most effective when collected 3-6 days after confluency and added at 50% to the culture medium. The CM-treated RPC-C2A cells showed high alkaline phosphatase activity, a high mRNA level of osteocalcin and decreases in the mRNA levels of osteopontin and osteonectin, but undetectable levels of mRNA of dentin sialophosphoprotein by Northern blot analyses. A pan-specific anti-transforming growth factor (TGF)-beta antibody and a soluble form of receptor for bone morphogenetic protein (BMP)-2/-4 did not neutralize the CM-induced mineralization. These results suggest that some soluble factor(s) other than TGF-beta or BMP-2/-4 in the CM from MC3T3-E1 cells cause differentiation of RPC-C2A cells to osteoblast-like cells.

Alkaline Phosphatase↗

PubMed2000

Effects of orbital spaceflight on human osteoblastic cell physiology and gene expression.

During long-term spaceflight, astronauts lose bone, in part due to a reduction in bone formation. It is not clear, however, whether the force imparted by gravity has direct effects on bone cells. To examine the response of bone forming cells to weightlessness, human fetal osteoblastic (hFOB) cells were cultured during the 17 day STS-80 space shuttle mission. Fractions of conditioned media were collected during flight and shortly after landing for analyses of glucose utilization and accumulation of type I collagen and prostaglandin E(2) (PGE(2)). Total cellular RNA was isolated from flight and ground control cultures after landing. Measurement of glucose levels in conditioned media indicated that glucose utilization occurred at a similar rate in flight and ground control cultures. Furthermore, the levels of type I collagen and PGE(2) accumulation in the flight and control conditioned media were indistinguishable. The steady-state levels of osteonectin, alkaline phosphatase, and osteocalcin messenger RNA (mRNA) were not significantly changed following spaceflight. Gene-specific reductions in mRNA levels for cytokines and skeletal growth factors were detected in the flight cultures using RNase protection assays. Steady-state mRNA levels for interleukin (IL)-1alpha and IL-6 were decreased 8 h following the flight and returned to control levels at 24 h postflight. Also, transforming growth factor (TGF)-beta(2) and TGF-beta(1) message levels were modestly reduced at 8 h and 24 h postflight, although the change was not statistically significant at 8 h. These data suggest that spaceflight did not significantly affect hFOB cell proliferation, expression of type I collagen, or PGE(2) production, further suggesting that the removal of osteoblastic cells from the context of the bone tissue results in a reduced ability to respond to weightlessness. However, spaceflight followed by return to earth significantly impacted the expression of cytokines and skeletal growth factors, which have been implicated as mediators of the bone remodeling cycle. It is not yet clear whether these latter changes were due to weightlessness or to the transient increase in loading resulting from reentry.

Alkaline Phosphatase↗

PubMed2002

Ovariectomy hinders the early stage of bone-implant integration: histomorphometric, biomechanical, and molecular analyses.

Postmenopausal osteoporosis is a contributing factor to alveolar bone atrophy associated with tooth loss in the elderly. The use of dental titanium implants has been increasingly adapted to treat these edentulous patients. This study examines whether female gonadal hormone deficiency interferes with the critical integration process between bone and implants. Two types of experimental titanium implants with acid-treated surfaces were placed in the femurs of ovariectomized (ovx) and sham-operated control rats: T-cell implants with a hollow chamber for histomorphometric and steady-state mRNA expression assays, and unthreaded cylindrical implants for biomechanical push-in tests. At week 2, less bone area was found in the ovx-implant group (p = 0.0495) than in the sham-implant group. The implant push-in test showed that the ovx-implant group had approximately half of the withstanding value of the sham-implant group (p = 0.009). However, these differences between the ovx and sham groups became diminished at week 4. Total RNA samples were examined by a reverse transcriptase-polymerase chain reaction assay for col1a1, col3a1, bone sialoprotein (bSP) II, osteonectin, osteopontin, osteocalcin, integrin beta1 and integrin beta3. In untreated bones and in created bone defects without implant placement, ovx did not affect the steady-state levels of the mRNAs tested. When implants were placed, significant upregulation of these genes was observed in the sham-implant group; however, only osteocalcin and integrins were upregulated in the ovx-implant group. The results suggest a biphasic effect of female gonadal hormone deficiency that may temporarily interfere with the early implant-tissue integration process, and which may be associated with a failure to upregulate a selected set of bone extracellular matrix genes. Once established, however, functional bone-implant integration can be achieved even in ovx rats.

Animals↗

PubMed2002

2-methoxyestradiol induces interferon gene expression and apoptosis in osteosarcoma cells.

2-Methoxyestradiol (2-ME), a naturally occurring mammalian metabolite of 17beta-estradiol, has been implicated as a physiological inhibitor of tumor cell proliferation. In this study, the effects of 2-ME on cultured osteosarcomatous cells were investigated. Dose-dependent growth inhibition was observed in MG63 and TE85 human osteosarcoma cells exposed to 2-ME. The cell killing by 2-ME was ligand-specific; the immediate precursor (2-hydroxyestradiol), the parent compound (17beta-estradiol), and the equivalent metabolite of estrone (2-methoxyestrone) exhibited less potency and efficacy. Furthermore, 2-ME was similarly effective at killing immortalized human fetal osteoblastic cells (hFOB) with and without estrogen receptor-alpha and -beta and rat osteosarcoma cells (ROS17/2.8). The cytotoxicity of 2-ME was selective to transformed and immortalized osteoblastic cells; 2-ME (2 microm) had no effect on the proliferation of primary cultures of human osteoblasts. Co-treatment with the potent estrogen receptor ligand, ICI-182,780, did not reduce 2-ME-induced osteosarcoma cell death, implying that this action is not mediated by conventional estrogen receptors. The expression levels of bone matrix protein genes, type 1 collagen and osteonectin, were transiently reduced after 2-ME treatment, suggesting that the surviving cells are capable of producing bone matrix. The 2-ME-mediated killing of osteosarcoma cells was due to the induction of apoptosis; treatment induced expression of interferon genes within 12 h and histological evidence of apoptosis within 48 h of 2-ME treatment. Thus, our results demonstrate that 2-ME is highly cytotoxic to osteosarcoma cells but not normal osteoblasts. These findings suggest that further study of 2-ME as a potential intervention for treatment of osteosarcoma is warranted.

2-Methoxyestradiol↗

PubMed2002

Three-dimensional cultures of normal human osteoblasts: proliferation and differentiation potential in vitro and upon ectopic implantation in nude mice.

We report the establishment in vitro of three-dimensional (3D) cultures of human osteoblasts (hOB) derived from normal adults and supported uniquely by the extracellular matrix (ECM) they deposit. Osteoblasts were cultured in 3D cultures in vitro for up to 120 days. The 3D cultures, examined at 25, 31, and 48 days, expressed protein markers of osteoblastic cells, namely osteonectin, collagen type I, fibronectin, osteopontin, bone sialoprotein, biglycan, and decorin. Sequentially, alkaline phosphatase (AP) and then Ca incorporation, mineralization of matrix (monitored by histochemistry and transmission electron microscopy), and finally osteocalcin expression, were detected in the 3D cultures. Ultrastructurally, morphology progressed from early to mature osteoblast and to osteocyte-like. Cells were embedded in a matrix with organized collagen type I fibers containing, increasingly with time of culture, needle-shaped crystals, often associated with matrix vesicles, characteristic of those in bone. During the culture (up to 120 days) there was an outgrowth of proliferating osteogenic cells from the 3D structure. Subcutaneous implantation in nude mice for 20 days of osteoblasts cultured in 3D culture for different lengths of time in vitro, showed progression of mineralization from the inner region of the implant outward, with peripheral cells being embedded in nonmineralized, collagen-rich matrix. The 3D implants were invaded by vessels derived from the host.

Adult↗

PubMed2003

Modification of gene expression induced in human osteogenic and osteosarcoma cells by culture on a biphasic calcium phosphate bone substitute.

Bone hybrids made of bioceramics seeded with mesenchymal or osteoblastic cells are very promising alternatives to autologous bone graft. Along this line, the development of in vitro models, dedicated to analyze the influence of these biomaterials on osteogenic cells, will help to improve the performance of these bone substitutes. In the present work we analyzed the effects of a macroporous biphasic calcium phosphate ceramic (BCP, Triosite) on three different human osteosarcoma cell lines and on human primary osteogenic cells and compared this culture substratum to traditional culture on plastic. We showed that all these osteoblastic cells adhere and proliferate on the trabecular BCP blocks, with a different spatial organization for osteosarcoma cells compared to normal osteogenic cells. We also demonstrated that osteoblastic marker genes such as Cbfa1, type I collagen, osteonectin, osteopontin, and osteocalcin were expressed at similar levels by these cells cultured on either substratum, suggesting that adhesion to BCP does maintain the osteoblastic phenotype of these cells. Next, we provided the first evidence of differences of cytokine expression profiles revealed on this Ca-P ceramic as compared to expression in classical culture. These modifications affected the expression of cytokines such as TGF-beta1, G-CSF, and IL-3 and were quantitatively different between osteosarcoma cells and normal osteogenic cells. Given the role of these cytokines in bone biology and in hematopoiesis, these results obtained in vitro suggest that the BCP ceramic studied here could stimulate osteogenesis in vivo by activating cellular processes during bone formation and healing. This study highlights the notion that the nature of the culture substratum must be taken into account when studying bone cell biology in vitro. Owing to the nature and spatial organization of the BCP, our hypothesis is that culture on BCP is closer to the physiological situation than culture on plastic.

Adolescent↗

PubMed2000

Development of soluble glasses for biomedical use Part II: the biological response of human osteoblast cell lines to phosphate-based soluble glasses.

Soluble glasses are considered to be of potential clinical value in orthopaedic and dental surgery. However, the biological response to these materials is not well understood. To determine the effects of these glasses, two human osteoblast cell lines, MG63 and HOS (TE85), were incubated in vitro in the presence of increasing concentrations of extracts of the glasses. The effects of the extracts on cell growth was measured using the MTT assay and an ELISA assay was used to measure the expression of bone sialoprotein (BSP), osteonectin (ON) and fibronectin (FN), antigens which play a fundamental part in the integrity and function of hard connective tissue. The results showed that the proliferation of the cells was adversely affected only by the more soluble glasses, which also down-regulated the expression of the bone-associated proteins. In contrast, the extract of the glass with the lowest dissolution rate, which contains relatively elevated levels of Ca2+, was found to enhance bone cell growth and antigen expression. These findings suggest that the compositions of these glasses at least partly determine the response of cells and thus, that the glasses could be modified to elicit a more optimal biological response and clinical efficacy.

Journal Article↗

PubMed1997

Responses of bone to titania-hydroxyapatite composite and nacreous implants: a preliminary comparison by in situ hybridization.

The effect of two biomaterials on bone formation in vivo by in situ hybridization, was compared by using RNA probes complementary to collagen alpha1(I) RNA, osteonectin RNA and osteocalcin RNA. Holes were drilled into the midshafts of rat femurs. Titania-hydroxyapatite composite (THA) or nacre cylinders were implanted and the bone-implant regions collected 14 days after operation. Cuboidal osteoblasts, intensely labelled with the three probes, were seen to be lining the newly formed bone surrounding the THA implant. Between the implant and the new bone, a layer of un-labelled, apparently non-osteogenic cells was observed. By contrast, the nacre implant was bonded to the newly formed bone without any soft tissue interference. Osteoblasts lining the distal surface of the newly formed bone were stained with all three RNA probes, although weaker than in the THA sample. Some of the osteoblasts were flattened. We concluded from the appearance of the osteoblasts that the bone formation in the nacre samples had progressed beyond the phase of maximal synthetic activity. Around the THA implant, the labelling indicated that bone-forming activity was still high. It was concluded that the bioactivity of nacre was higher than that of THA.

Journal Article↗

PubMed2004

Adult stem cell driven genesis of human-shaped articular condyle.

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivo implantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitro experiments using goat and rat MSCs corroborated in vivo data by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivo incubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.

Animals↗