Effects of titanium surface roughness on mesenchymal stem cell commitment and differentiation signaling.

PURPOSE: Human mesenchymal stem cells (hMSCs) are primary cells capable of differentiating to osteocytic lineage when stimulated under appropriate conditions. This study examined changes in hMSC morphology, proliferation, and gene expression after growth on machined or dual acid-etched (AE) titanium surfaces. MATERIALS AND METHODS: hMSCs, isolated from adult human bone marrow, were cultured on titanium surfaces. The two specimens of titanium surfaces in this study included machined and AE titanium disks. Cell morphology was evaluated by scanning electron microscopy, and cell proliferation and collagen synthesis were estimated by measuring the amount of 3H-thymidine incorporation into DNA and 3H-proline incorporation into collagen fibers. Alkaline phosphatase (ALP) activity was determined by measuring the release of p-nitrophenol from disodium p-nitrophenyl phosphate. Changes in gene expression for bone morphogenetic protein-2 (BMP-2), Runx2 type II, Osterix (Osx), osteopontin, type I collagen, ALP, osteocalcin, and bone sialoprotein were determined by reverse-transcriptase polymerase chain reaction after 22 days of in vitro culture in osteogenic medium. RESULTS: The two substrates had no significant effects on cell adhesion and proliferation. Morphologic characteristics were observed by scanning electron microscopy. hMSCs on the machined surface spread more and were flatter than cells cultured on the AE surface. Osteopontin mRNA expression was similar on all surfaces, and the other mRNA transcripts were increased in hMSC cultured on AE surface. In particular, BMP-2, Runx2, and Osx, three osteogenic factors that induce the progressive differentiation of multipotent mesenchymal cells into osteoblasts, were expressed more on AE titanium than on machined titanium. Collagen and ALP assays confirmed the highest level of mRNA transcripts correlated with increases in these proteins. CONCLUSION: These results showed that an AE titanium surface stimulated the expression of markers of osteoblastic phenotype more than a machined titanium surface.

Encapsulation, in vitro characterization, and in vivo biocompatibility of Sertoli cells in alginate-based microcapsules.

A method for microencapsulation of isolated neonatal porcine Sertoli cells is described. Using a conventional alginate-poli-L-ornithine encapsulation procedure, which has been used in our laboratory for almost two decades to envelop pancreatic islets, we observed significant loss of Sertoli cell viability, possibly due to excessive Ca(2+) ion exposure. Replacing calcium with barium, or shortening the incubation period in the presence of Ca ions, we obtained barium or calcium alginate gel microbeads that did not alter morphology and viability of the encapsulated Sertoli cells. The procedure might permit access to a novel approach to immunologically alter cell graft acceptance.

Effect of titanium surface roughness on human osteoblast proliferation and gene expression in vitro.

PURPOSE: Cell proliferation and extracellular matrix formation are primary events in bone formation. At the dental implant-tissue interface, implant surface roughness modulates osteoblast functions. The aim of the present in vitro study was to investigate the effect of varying surface roughness of titanium implant material on cell proliferation and mRNA expression of specific markers of osteoblast phenotype. MATERIALS AND METHODS: Primary cultures of osteoblasts derived from human mandibular bone were cultured on titanium surfaces. Three titanium surfaces were studied: machined titanium, microsandblasted titanium, and macro-sandblasted titanium (average surface roughnesses of 0.5 and 3 microm, respectively). Cell morphology was estimated by scanning electron microscope analysis and cell proliferation by measuring the amount of 3H-thymidine incorporation into DNA. mRNA expression of osteonectin, osteopontin, bone sialoprotein (BSP), and Runx2, which are markers of osteoblastic phenotype, were determined by reverse transcriptase polymerase chain reaction (RT-PCR) analysis. RESULTS: Human osteoblasts cultured on machined titanium spread more and were flatter than cells cultured on rough titanium. All blasted surfaces showed significantly higher DNA synthesis than the machined surfaces. Osteonectin mRNA expression was similar on all surfaces. Other mRNA transcripts were increased in osteoblasts cultured on rough titanium surfaces, particularly the macrosandblasted surface. CONCLUSIONS: An average surface roughness of 3 microm (macro-sandblasted titanium) is more suitable than an average surface roughness of 0.5 microm (micro-sandblasted titanium) in favoring osteoblast differentiation in vitro.

Multipotent mesenchymal stem cells with immunosuppressive activity can be easily isolated from dental pulp.

BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) are currently being investigated in preclinical and clinical settings because of their multipotent differentiative capacity or, alternatively, their immunosuppressive function. The aim of this study was to evaluate dental pulp (DP) as a potential source of MSCs instead of bone marrow (BM). METHODS: Flow cytometric analysis showed that DP-MSCs and BM-MSCs were equally SH2, SH3, SH4, CD29 and CD 166 positive. The in vitro proliferative kinetics of MSCs were measured by 3H-thymidine incorporation uptake. The immunosuppressive function of MSCs was then tested by coculturing PHA-stimulated allogeneic T cells with or without MSCs for 3 days. RESULTS: BM-MSCs could be differentiated in vitro into osteogenic, chondrogenic and adipogenic lineages. DP-MSCs showed osteogenic and adipocytic differentiation, but did not differentiate into chondrocytes. Although DP-MSCs grow rapidly in vitro between day 3 and day 8 of culture and then decrease their proliferation by day 15, BM-MSCs have a stable and continuous proliferation over the same period of time. The addition of DP-MSCs or BM-MSCs resulted in 91 +/- 4% and 75 +/- 3% inhibition of T cell response, respectively, assessed by a 3H-thymidine assay. CONCLUSIONS: Dental pulp is an easily accessible and efficient source of MSCs, with different kinetics and differentiation potentialities from MSCs as isolated from the bone marrow. The rapid proliferative capacity together with the immunoregulatory characteristics of DP-MSCs may prompt future studies aimed at using these cells in the treatment or prevention of T-cell alloreactivity in hematopoietic or solid organ allogeneic transplantation.

Accelerated functional maturation of isolated neonatal porcine cell clusters: in vitro and in vivo results in NOD mice.

Neonatal porcine cell clusters (NPCCs) might replace human for transplant in patients with type 1 diabetes mellitus (T1DM). However, these islets are not immediately functional, due to their incomplete maturation/ differentiation. We then have addressed: 1) to assess whether in vitro coculture of islets with homologous Sertoli cells (SC) would shorten NPCCs' functional time lag, by accelerating the beta-cell biological maturation/differentiation; 2) to evaluate metabolic outcome of the SC preincubated, and microencapsulated NPCCs, upon graft into spontaneously diabetic NOD mice. The islets, isolated from < 3 day piglets, were examined in terms of morphology/viability/function and final yield. SC effects on the islet maturation pathways, both in vitro and in vivo, upon microencapsulation in alginate/poly-L-ornithine, and intraperitoneal graft into spontaneously diabetic NOD mice were determined. Double fluorescence immunolabeling showed increase in beta-cell mass for SC+ neonatal porcine islets versus islets alone. In vitro insulin release in response to glucose, as well as mRNA insulin expression, were significantly higher for SC+ neonatal porcine islets compared with control, thereby confirming SC-induced increase in viable and functional beta-cell mass. Graft of microencapsulated SC+ neonatal porcine islets versus encapsulated islets alone resulted in significantly longer remission of hyperglycemia in NOD mice. We have preliminarily shown that the in vitro NPCCs' maturation time lag can dramatically be curtailed by coincubating these islets with SC. Graft of microencapsulated neonatal porcine islets, precultured in Sertoli cells, has been proven successful in correcting hyperglycemia in stringent animal model of spontaneous diabetes.

Biocompatibility of collagen membranes crosslinked with glutaraldehyde or diphenylphosphoryl azide: an in vitro study.

Crosslinking of collagen biomaterials increases their resistance to degradation in vivo. Glutaraldehyde (GA) is normally used to crosslink collagen biomaterial, but is often cytotoxic. Diphenylphosphoryl azide (DPPA) has recently been proposed as reagent, but little is known about its effects on cell behavior. In this study, we determined which collagen membrane was the most biocompatible: Paroguide which is crosslinked with DPPA and contains chondroitin sulfate; Opocrin which is crosslinked with DPPA; Biomed Extend which is crosslinked with GA; and Bio-Gide which is left untreated. Cell proliferation and extracellular matrix macromolecule deposition were evaluated in human fibroblasts cultured on the membranes. The GA-crosslinked Biomed Extend membrane and the not-crosslinked Bio-Gide membrane reduced cell growth and collagen secretion compared with DPPA-crosslinked biomembranes. When Paroguide and Opocrin were compared, better results were obtained with Paroguide. The greatest amount of transforming growth factor beta1, a growth factor involved in extracellular matrix macromolecule accumulation and in tissue regeneration, was produced by cells cultured on Paroguide, with Opocrin second. Our data suggest that the DPPA method is more biocompatible than the GA for crosslinking collagen biomaterials and that membranes made of collagen plus chondroitin sulfate are better than membranes made of pure collagen.

Conformal polymer coatings for pancreatic islets transplantation.

The aim of this work was to improve an aqueous two-phase system methodology for fabrication of coherent microcapsules. Simulated microgravity was investigated as tool to improve the cell cluster morphology in order to increase the overall quality of conformal polymer coatings, while the application of two concentric alginate layers and the use of barium instead of calcium as gelling ion was evaluated. Simulated microgravity enabled improvement of neonatal porcine cell cluster sphericity however the freely floating cells, originated during incubation and often found on the capsule surface, raised immunological concerns. Overall, these technical changes translated into improving quality of microcapsules, in terms of either morphologic aspects or the membrane's functional performance. Preparation procedure did not seem to adversely affect viability of the embodied cells. Moreover, the employed alginates high biocompatibility, per se, would promote a good encapsulated cell engraftment. Minimization of last generation microcapsule's size, made of highly purified alginates, represents a further advance on the new horizons of cell therapy for the treatment of a wide variety of chronic disorders, including insulin-dependent diabetes mellitus.

Effects of hydroxyapatite and Biostite on osteogenic induction of hMSC.

When isolated from the iliac crest human mesenchymal stem cells (hMSC) differentiate into osteoblast-like cells with appropriate stimulation in culture. This in vitro study tested the hypothesis that Biostite and hydroxyapatite (HA) affect proliferation and differentiation of hMSC into osteoblastic cells. Cell proliferation was determined by measuring 3H-thymidine incorporation into DNA and typical markers of osteoblastic phenotype were determined by RT-PCR assay. No differences emerged in cell proliferation cultures with Biostite or hydroxyapatite (HA), but gene expression analysis revealed higher expression of collagen,alkaline phosphatase (ALP), osteopontin and bone sialoprotein (BSP) in the presence of Biostite. TGFb2 production, as assessed by an Elisa kit, and Runx2 expression by RT-PCR, were greater in Biostite cultures, suggesting Biostite provides a better environment for hMSC differentiation into osteoblasts and is, potentially, a more promising bone-filling material than HA.