Repair of critical size defects in the rat cranium using ceramic-reinforced PLA scaffolds obtained by supercritical gas foaming

Bioresorbable scaffolds made Of poly(L-lactic acid) (PLA) obtained by supercritical gas foaming were recently described as suitable for tissue engineering, portraying biocompatibility with primary osteoblasts in vitro and interesting mechanical properties when reinforced with ceramics. The behavior of such constructs remained to be evaluated in vivo and therefore the present study was undertaken to compare different PLA/ceramic composite scaffolds obtained by supercritical gas foaming in a critical size defect craniotomy model in Sprague-Dawley rats. The host-tissue reaction to the implants was evaluated semiquantitatively and similar tendencies were noted for all graft substitutes: initially highly reactive but decreasing with time implanted. Complete bone-bridging was observed IS weeks after implantation with PLA/ 5 wt % beta-TCP (PLA/TCP) and PLA/5 wt % HA (PLA/HA) scaffolds as assessed by histology and radiography. We show here for the first time that this solvent-free technique provides a promising approach in tissue engineering demonstrating both the biocompatibility and osteoconcluctivity of the processed structures in vivo

Fetal bone cells for tissue engineering

We envision the use of human fetal bone cells for engineered regeneration of adult skeletal tissue. A description of their cellular function is then necessary. To our knowledge, there is no description of human primary fetal bone cells treated with differentiation factors. The characterization of fetal bone cells is particularly important as the pattern of secreted proteins from osteoblasts has been shown to change during aging. In the first part of this work, human primary fetal bone cells were compared to adult bone cells and mesenchymal stem cells for their ability to proliferate and to differentiate into osteoblasts in vitro. Cell proliferation, gene expression of bone markers, alkaline phosphatase (ALP) activity, and mineralization were analyzed during a time-course study. In the second part of this paper, bone fetal cells behavior exposed to osteogenic factors is further detailed. The doubling time of fetal bone cells was comparable to mesenchymal stem cells but significantly shorter than for adult bone cells. Gene expression of cbfa-1, ALP, alpha1 chain of type I collagen, and osteocalcin were upregulated in fetal bone cells after 12 days of treatment, with higher inductions than for adult and mesenchymal stem cells. The increase of ALP enzymatic activity was stronger for fetal than for adult bone cells reaching a maximum at day 10, but lower than for mesenchymal stem cells. Importantly, the mineralization process of bone fetal cells started earlier than adult bone and mesenchymal stem cells. Proliferation of fetal and adult bone cells was increased by dexamethasone, whereas 1alpha,25-dihydroxyvitamin D3 did not show any proliferative effect. Mineralization studies clearly demonstrated the presence of calcium deposits in the extracellular matrix of fetal bone cells. Nodule formation and calcification were strongly increased by the differentiation treatment, especially by dexamethasone. This study shows for the first time that human primary fetal bone cells could be of great interest for bone research, due to their fast growth rate and their ability to differentiate into mature osteoblasts. They represent an interesting and promising potential for therapeutic use in bone tissue engineering

Bioresorbable composites prepared by supercritical fluid foaming

Bone is a natural composite construct, with a gradient structure going from a loose interconnected cellular core to an outer dense wall, thus minimizing bone weight while keeping a high mechanical resistance. Due to this unique and complex structure, bone defects are difficult to replace or repair. Tissue engineering aims at providing artificial bone grafts. Several techniques have been proposed to produce porous structures or scaffolds, but, as yet, with no optimal solutions. This article focuses on bioresorbable ceramic-polymer composite foams obtained by supercritical fluid foaming. This flexible technique enables an adequate morphology and suitable properties for bone tissue engineering to be obtained. Composite scaffolds are biocompatible, allowing cell proliferation and differentiation

A composite biofoam, from research to in vivo validation

Bone tissue engineering aims at developing scaffolds, or porous structures, as supports for cell migration, proliferation and differentiation, in order to favor bone healing. L-poly lactic acid (PLA) foams, which were reinforced or not with β-tricalcium phosphate (β-TCP) particles, were obtained by melt-extrusion followed by supercritical gas foaming. After tailoring their morphology and mechanical properties to those of natural cancellous bone, their in vitro biocompatibility was tested. Promising results led to in vivo studies, using calvarial critical size defect in rats and bone filling of tibial and femoral defects in shee

Repair of critical size defects in the rat cranium using ceramic-reinforced PLA scaffolds obtained by supercritical gas foaming

Bioresorbable scaffolds made of poly(L-lactic acid) (PLA) obtained by supercritical gas foaming were recently described as suitable for tissue engineering, portraying biocompatibility with primary osteoblasts in vitro and interesting mechanical properties when reinforced with ceramics. The behavior of such constructs remained to be evaluated in vivo and therefore the present study was undertaken to compare different PLA/ceramic composite scaffolds obtained by supercritical gas foaming in a critical size defect craniotomy model in Sprague-Dawley rats. The host-tissue reaction to the implants was evaluated semiquantitatively and similar tendencies were noted for all graft substitutes: initially highly reactive but decreasing with time implanted. Complete bone-bridging was observed 18 weeks after implantation with PLA/ 5 wt % beta-TCP (PLA/TCP) and PLA/5 wt % HA (PLA/HA) scaffolds as assessed by histology and radiography. We show here for the first time that this solvent-free technique provides a promising approach in tissue engineering demonstrating both the biocompatibility and osteoconductivity of the processed structures in vivo

Biocompatibility of bioresorbable poly(L-lactic acid) composite scaffolds obtained by supercritical gas foaming with human fetal bone cells

The aim of this investigation was to test the biocompatibility of three-dimensional bioresorbable foams made of poly(L-lactic acid) (PLA), alone or filled with hydroxyapatite (HA) or beta-tricalcium phosphate (beta-TCP), with human primary osteoblasts, using a direct contact method. Porous constructs were processed by supercritical gas foaming, after a melt-extrusion of ceramic/polymer mixture. Three neat polymer foams, with pore sizes of 170, 310, and 600 microm, and two composite foams, PLA/5 wt% HA and PLA/5 wt% beta-TCP, were examined over a 4-week culture period. The targeted application is the bone tissue-engineering field. For this purpose, human fetal and adult bone cells were chosen because of their highly osteogenic potential. The association of fetal bone cells and composite scaffold should lead to in vitro bone formation. The polymer and composite foams supported adhesion and intense proliferation of seeded cells, as revealed by scanning electron microscopy. Cell differentiation toward osteoblasts was demonstrated by alkaline phosphatase (ALP) enzymatic activity, gamma-carboxylated Gla-osteocalcin production, and the onset of mineralization. The addition of HA or beta-TCP resulted in higher ALP enzymatic activity for fetal bone cells and a stronger production of Gla-osteocalcin for adult bone cells

Epigenetic regulation during fetal femur development: DNA methylation matters

Epigenetic modifications are heritable changes in gene expression without changes in DNA sequence. DNA methylation has been implicated in the control of several cellular processes including differentiation, gene regulation, development, genomic imprinting and X-chromosome inactivation. Methylated cytosine residues at CpG dinucleotides are commonly associated with gene repression; conversely, strategic loss of methylation during development could lead to activation of lineage-specific genes. Evidence is emerging that bone development and growth are programmed; although, interestingly, bone is constantly remodelled throughout life. Using human embryonic stem cells, human fetal bone cells (HFBCs), adult chondrocytes and STRO-1+ marrow stromal cells from human bone marrow, we have examined a spectrum of developmental stages of femur development and the role of DNA methylation therein. Using pyrosequencing methodology we analysed the status of methylation of genes implicated in bone biology; furthermore, we correlated these methylation levels with gene expression levels using qRT-PCR and protein distribution during fetal development evaluated using immunohistochemistry. We found that during fetal femur development DNA methylation inversely correlates with expression of genes including iNOS (NOS2) and COL9A1, but not catabolic genes including MMP13 and IL1B. Furthermore, significant demethylation was evident in the osteocalcin promoter between the fetal and adult developmental stages. Increased TET1 expression and decreased expression of DNA (cytosine-5-)-methyltransferase 1 (DNMT1) in adult chondrocytes compared to HFBCs could contribute to the loss of methylation observed during fetal development. HFBC multipotency confirms these cells to be an ideal developmental system for investigation of DNA methylation regulation. In conclusion, these findings demonstrate the role of epigenetic regulation, specifically DNA methylation, in bone development, informing and opening new possibilities in development of strategies for bone repair/tissue engineering.<br/