We have used human Embryonic Stem cells (hESC) and human Mesenchymal Stem Cells (hMSC) in rat models of bone repair in order to assess the efficacy of these cells for treatments of trauma and skeletal diseases. Graft survival is considered to be of key importance to efficacy of these treatments. Therefore the aim of this study was to develop a technique for identifying implanted cells in histological preparations without the need for genetic engineering of the implanted cells.

Methods: In our experiments hES and hMSC were pre-differentiated during cell culture towards the osteoblast lineage, and then implanted in a Demineralised Bone Matrix (DBM) carrier into an experimentally created full thickness calvarial bone lesion. The animals were sampled seven days and fourteen days after implantation into either immune deficient (RNU-Foxn1rnu) or immune competent (wild type) Sprague Dawley rats. Fluorescent In Situ Hybridisation (FISH) using whole human genome probes identified the human cells within the host lesion site.

Results: Our results have demonstrated that hESC and hMSC derived cells survive in both immune competent (wild type) and immune compromised (nude) animals for the initial seven days post implantation. On the other hand while both the hESC and hMSC derived cells are capable of surviving for at least 14 days in immune compromised animals they do not survive for this period of time in immune competent animals.

Discussion: It appears that the cell/DBM graft is not rejected within seven days even when exposed to the wild type hosts T cell response. However longer term survival required an immune deficient model that is lacking in a T cell response. This data points to interesting future studies regarding which components of the host response are responsible for xenogenic stem cell implant rejection.

The use of stem cells in tissue engineering has emerged as a promising therapy for the repair of bone and cartilage defects. Targeted delivery of stem cells requires a substrate to maintain the cells at the repair site, as well as to provide the physical cues, such as mechanical strain, for encouraging differentiation and expression of the mature cell phenotype. The strains that will be generated in cells residing on the scaffold is dependent on the scaffold material, as well as both the fibre thickness and the fibre orientation in the scaffold. To encourage uniform bone matrix generation throughout the scaffold, it is desirable that the strain be uniformly distributed and that the internal pore architecture be precisely controlled to maximise media diffusion. This requires an optimised scaffold design and a manufacturing technique that allows for precise control over the scaffold’s internal architecture.

Scaffold architecture was optimised by performing a series of finite element analyses (FEA) on computer aided design (CAD) models of Polycaprolactone (PCL) scaffolds. The mechanical properties of PCL were used to yield an accurate strain profile of scaffolds with different fibre orientations. Having determined the optimal scaffold geometry, PCL scaffolds were manufactured using a fibre deposition technique that yielded three-dimensional objects with this geometry. During manufacture, a PCL solution was extruded into a non-miscible solvent which precipitated out PCL fibres in repetitive layers. Of the geometries tested with FEA, a 90 degree rotation of adjacent layers with a 50% offset of parallel strands was found to provide the optimal strain distribution (60% increase in surface exposed to strain). Histomorphometry was used to assess the exact dimensions of the scaffold produced. Fibre spacing was found to be precisely controlled to 380 +/- 10 microns within the layers and the fibre thickness was controlled to 270 +/- 10 microns.

This demonstrates that FEA can be used to predict the strain distribution of different CAD models and that the fibre deposition solvent extrusion technique can be used to accurately manufacture PCL scaffolds that match the desired architecture.

Aim: To investigate the directed chondrogenic differentiation of human embryonic and adult stem cells in 3D alginate bead culture.

Introduction: Cartilage possesses limited self-renewal potential and current repair of damage due to trauma or disease involves removal of non-load bearing chon-drocytes from a healthy part of the joint, expansion of chondrocytes and subsequent surgery to replace damaged, load-bearing cartilage. We investigated the potential of human embryonic and adult stem cells as an alternative cell source for cartilage repair.

Experimental design: Human embryonic stem cells (hESC) and human adult marrow stromal cells (hMSCs) cells were cultured in alginate in a 3D bead format in control or chondrogenic media over a 21day period. Cells were subsequently released from their matrix for gene expression analysis or fixed within alginate beads and crytostat sections prepared for immunostaining and histology.

Cell types used: H9 human embryonic stem cells, bone-marrow derived hMSCs and HEK293 (human embryonic kidney epithelium cell line, used as a negative control).

Data: H9 and hMSC cells cultured in alginate beads bathed in control media have a denser matrix with no lacunae-like structures compared to those cultured in the presence of chondrogenic media. The presence of chondrogenic media results in a matrix containing cells within lacunae-like structures very similar to those seen in human cartilage. In contrast, HEK293 cells formed large highly cellular clusters which had clearly undergone significant proliferation. As both H9 and HEK293 cells are highly proliferative the reduction in the proliferative potential of the chondrogenic H9 derived cells is consistent with entry into a stable terminally differentiated state.

Immunostaining demonstrated that hMSCs and H9 cells express cartilage specific Collagen II and Collagen X.

Conclusion: 3D culture of adult hMSCs and hESC (H9) in alginate beads has resulted in stable directed differentiation down the chondrogenic lineage. These data point towards the future use of these human cell sources in cartilage repair.

Introduction: Several recent studies have highlighted the influence of topographical features on the response of cells to biomaterial surfaces, both in terms of their adhesion, morphology and gene expression. Initial cell adhesion events are believed to be pivotal in dictating subsequent host response to implant materials and therefore understanding the mechansims that regulate these events is fundemental to the design and engineering of the next generation of biomaterials. In our studies we evaluated the adhesion associated events of osteoblasts on four orthopaedic metals, each produced to the same surface finnish. Scanning Electron Microscopy (SEM) and Atomic Force Microscopy (AFM) were used to determine the nanometre scale topography and immunofluorescence microscopy and image analysis performed to evaluate cell morphology.

Methods: Vitallium, titanium grade 2 (Ti2), Ti6Al4V and TM2F discs were prepared by Stryker, machined and finished to 1 micron. SEM and AFM were then used to analyse surface topography. Rat primary osteoblasts were then seeded at low density onto the metal discs and allowed to adhere and spread for 24 hours. The cells where fixed and focal adhesions stained with an anti-vinculin Mab. The actin cytoskeleton was counterstained with TRITC phalloidin and nuclei stained with DAPI. Images where captured on both a standard epiflourescence microscope and a confocal microscope. Image analysis was performed using ScionImage^TM to determine cell area, major X/Y axis lengths and numbers of focal adhesions per cell.

Results: Gross observation of all samples revealed a perfectly smooth and flat surface. SEM and AFM analysis showed that at the nanometre scale each exhibited varying degrees of surface roughness. Vitallium was the smoothest with scratches a few nanometres deep running across the surface. In contrast Ti6Al4V, Ti2 and TM2F had increasing degrees of surface roughness, each with details that measured up to a few microns in height.

We measured 1: the area occupied by a cell and 2: the number of focal adhesions per cell. The largest values of osteoblastic cell area were seen with the smoother vitallium surface. In contrast, samples with more numerous and larger surface features resulted in the osteoblasts covering a smaller area and being confined by topographical elements (Ti2> TM2F> Ti6Al4V). In terms of adhesion, there were generally more focal adhesions per cell on rougher surfaces (Ti6Al4V> TM2F> Vitallium> Ti2).

Conclusions: The different nanometre scale features introduced through the manufacturing process of different orthopaedic implant materials influence the adhesion and cell morphology of osteoblast cells within the first 24 hours of contact. This may have consequences for later differentiation and function of these cells.

Introduction: Emerging therapies for regenerating skeletal tissues are focused on the repair of pathologically altered tissue by the transplantation of functionally competent cells and supportive matrices. Stem cells have the potential to differentiate into musculoskeletal tissue and may be the optimal cell source for such therapies. In vitro studies have demonstrated the ability of adult bone marrow stromal cells (MSC) and human embryonic stem cells (hES) to generate bone, but little is known regarding their potential to repair bone in vivo. Preclinical studies in animal models will allow investigation into the extent that regenerated tissue resembles functional and healthy tissue, and its potential clinical application.

Aim: To assess whether adult and embryonic stem cells maintained their ability to form musculoskeletal tissues in vivo using diffusion chambers implanted into the peritoneal cavity of nude mice. Currently, ongoing experiments are assessing the use of MSCs and hES cells to regenerate bone in a rodent preclinical model.

Methods: MSC cells and embryoid body-derived H9 hES cells were prepared as previously described (Haynesworth et al Bone 1992; Sottile et al Cloning Stem Cells 2003). Groups of cells were left untreated or pre-treated with osteogenic (OS) media for 5 days. Study 1: Single cell suspensions of untreated or pre-treated cells were injected into diffusion chambers which were implanted intraperitonealy into nude mice and left for 79 days. Study 2: OS pre-treated cells were implanted into an experimentally created full thickness calvarial defect in adult male Wistar rats. The defect area was left empty or filled with demineralised bone matrix (DBM: Allosource®) alone or with DBM/MSCs or DBM/hES composite. Tissues were collected 4 weeks after surgery.

Analysis: Histological and immunochemical techniques were used to evaluate cell phenotypes and the contribution of transplanted cells to tissue repair.

Results: Study 1: Both hES (in 2/3 chambers) and MSC (3/3) cells pre-treated with OS media formed only mineralised bone. No cartilage was detected in these OS pre-treated cells. Untreated hES cells formed both mineralised bone and cartilage within the chambers (2/3). In contrast, untreated MSC cells (3/3) produced no mineralised bone or cartilage. Preliminary analysis demonstrated the absence of any other tissue type in the diffusion chambers. Study 2: Active bone regeneration was observed at the edges of the calvarial defect after 4 weeks, with a high density of cells present within the MSC or hES/DBM composite. No signs of local cellular immunological response were seen.

Summary: OS pre-treatment restricted differentiation towards the osteoblast lineage in both hES and MSC cells indicating successful directed differentiation in vivo. Untreated hES and MSC cells produce a different range of cell phenotypes suggesting that the two cell sources represent cells at a different stage of commitment in a common cell lineage or cells derived from two distinct cell lineages. New bone formation was seen at the site of the calvarial defect in the presence OS pre-treated MSC and hES cells suggesting that these cells may support in vivo bone repair in a preclinical model. Funded by Geron Corporation