Development and characterization of a predictive microCT-based non-union model in Fischer F344 rats.

INTRODUCTION: Non-unions remain a clinical problem and are characterised by the failure to heal after a defined period of time. Current preclinical non-union models apply a wide variety of techniques to diminish intrinsic healing potential deviating from the clinical situation. The aim of this study was to develop and characterise a non-union model in rats using internal plate fixation without the need for additional healing insults, whereby bone healing can be longitudinally assessed using microCT. It was hypothesized that healing/non-unions can be accurately predicted at early time points by microCT. MATERIALS AND METHODS: Female, skeletally mature Fischer F344 rats received a 2 mm or 1 mm femoral osteotomy, stabilized with either a 2 mm thick plate or a 1.25 mm thick plate. Healing was monitored by microCT over 14 weeks and histological analysis at euthanasia. The mechanical environment was characterised using finite element (FE) modelling and biomechanical testing. RESULTS: The majority of animals receiving the 2 mm thick plate displayed poor healing responses in both the 2 mm and 1 mm defect size groups. Bone and cartilage formation were markedly improved using the 1.25 mm thick plate. MicroCT could accurately predict bone forming capacity at early time points (3-4 weeks). CONCLUSIONS: The 2 mm thick plating system confers poor healing responses in female Fischer F344 rats, comparable to atrophic non-unions. By reducing plate thickness to increase interfragmentary strain within the defect site healing is improved, leading to borderline healing situations or increased abundance of cartilage tissue present in the defect site with ultimate failure to bridge the defect (hypertrophic non-union). Furthermore, microCT can reliably identify delayed/non-healing animals within 4 weeks, thereby allowing their selective targeting for the testing of novel, clinically relevant treatment strategies in different clinical situations aimed at restoring impaired bone healing.

Tissue engineering and regenerative approaches to improving the healing of large bone defects.

Despite the high innate regenerative capacity of bone, large osseous defects fail to heal and remain a clinical challenge. Healing such defects requires the formation of large amounts of bone in an environment often rendered hostile to osteogenesis by damage to the surrounding soft tissues and vasculature. In recent years, there have been intensive research efforts directed towards tissue engineering and regenerative approaches designed to overcome this multifaceted challenge. In this paper, we describe and critically evaluate the state-of-the-art approaches to address the various components of this intricate problem. The discussion includes (i) the properties of synthetic and natural scaffolds, their use in conjunction with cell and growth factor delivery, (ii) their vascularisation, (iii) the potential of gene therapies and (iv) the role of the mechanical environment. In particular, we present a critical analysis of where the field stands, and how it can move forward in a coordinated fashion.

Pericyte plasticity - comparative investigation of the angiogenic and multilineage potential of pericytes from different human tissues.

Pericyte recruitment is essential for the stability of newly formed vessels. It was also suggested that pericytes represent common ancestor cells giving rise to mesenchymal stem cells (MSCs) in the adult. Here, we systematically investigated pericytes and MSCs from different human tissues in terms of their angiogenic and multilineage differentiation potential in vitro in order to assess the suitability of the different cell types for the regeneration of vascularised tissues. Magnetic-activated cell sorting (MACS®) was used to enrich CD34-CD146+ pericytes from adipose tissue (AT) and bone marrow (BM). The multilineage potential of pericytes was assessed by testing their capability to differentiate towards osteogenic, adipogenic and chondrogenic lineage in vitro. Pericytes and endothelial cells were co-seeded on Matrigel™ and the formation of tube-like structures was examined to study the angiogenic potential of pericytes. MSCs from AT and BM were used as controls. CD34-CD146+ cells were successfully enriched from AT and BM. Only BM-derived cells exhibited trilineage differentiation potential. AT-derived cells displayed poor chondrogenic differentiation upon stimulation with transforming growth factor-ß1. Interestingly, osteogenic differentiation was more efficient in AT-PC and BM-PC compared to the respective full MSC population. Matrigel™ assays revealed that pericytes from all tissues integrated into tube-like structures. We show that MACS®-enriched pericytes from BM and AT have the potential to regenerate tissues of different mesenchymal lineages and support neovascularisation. MACS® represents a simple enrichment strategy of cells, which is of particular interest for clinical application. Finally, our results suggest that the regenerative potential of pericytes depends on their tissue origin, which is an important consideration for future studies.

Cell therapy for bone repair: narrowing the gap between vision and practice.

This position paper summarises a vision of how cell-based therapies can be applied clinically to regenerate bone, as well as the steps needed to narrow the gap between that vision and clinical reality. It is a result of the presentations and discussion of the "Cell Therapy for Bone Repair" breakout session at the AO Foundation Symposium "Where Science Meets Clinics" in Davos, Switzerland from September 5-7, 2013. Participants included leaders from science, medicine, and industry from around the world. The session included clinical and scientific presentations, as well as an extended discussion among participants. Bone tissue has an innate regenerative capacity that in most cases allows functional healing at damage sites. However, there are a number of serious conditions in which bone does not fully heal and the result is significant morbidity. The clinical need for new therapies is clear, and the breakout session participants were enthusiastic about the potential impact on cell-based therapies for bone repair in the clinic. However, they also recognised the significant challenges that face the development of commercially viable cell therapy products. This paper outlines a vision in which patient selection is based on expected therapeutic outcome to create a consistently successful, cost-effective, cell-based therapy for bone repair. The need for a more complete understanding of bone repair, a better infrastructure for preclinical studies, and the need for collaboration among stakeholders is discussed.

Platelet-rich plasma induces annulus fibrosus cell proliferation and matrix production.

PURPOSE: Platelet-rich plasma (PRP) contains growth factors and creates a 3D structure upon clotting; PRP or platelet lysate (PL) might be considered for annulus fibrosus (AF) repair. METHODS: Bovine AF cells were cultured with 25% PRP, 50% PRP, 25% PL, 50% PL, or 10% FBS. After 2 and 4 days, DNA, glycosaminoglycan (GAG), and mRNA levels were analyzed. Histology was performed after injection of PRP into an AF defect in a whole disc ex vivo. RESULTS: By day 4, significant increases in DNA content were observed in all treatment groups. All groups also showed elevated GAG synthesis, with highest amounts at 50% PL. Collagen I and II expression was similar between groups; aggrecan, decorin, and versican expression was highest at 25% PL. Injection of PRP into the AF defect resulted in an increased matrix synthesis. CONCLUSIONS: Platelet-rich preparations increased the matrix production and cell number and may therefore be considered to promote AF repair.

3D scaffolds co-seeded with human endothelial progenitor and mesenchymal stem cells: evidence of prevascularisation within 7 days.

Blood supply is a critical issue in most tissue engineering approaches for large defect healing. As vessel ingrowth from surrounding tissues is proven to be insufficient, current strategies are focusing on the neo-vascularisation process. In the present study, we developed an in vitro pre-vascularised construct using 3D polyurethane (PU) scaffolds, based on the association of human Endothelial Progenitor Cells (EPC, CD34+ and CD133+) with human Mesenchymal Stem Cells (MSC). We showed the formation of luminal tubular structures in the co-seeded scaffolds as early as day 7 in culture. These tubular structures were proven positive for endothelial markers von Willebrand Factor and PECAM-1. Of special significance in our constructs is the presence of CD146-positive cells, as a part of the neovasculature scaffolding. These cells, coming from the mesenchymal stem cells population (MSC or EPC-depleted MSC), also expressed other markers of pericyte cells (NG2 and αSMA) that are known to play a pivotal function in the stabilisation of newly formed pre-vascular networks. In parallel, in co-cultures, osteogenic differentiation of MSCs occurred earlier when compared to MSCs monocultures, suggesting the close cooperation between the two cell populations. The presence of angiogenic factors (from autologous platelet lysates) in association with osteogenic factors seems to be crucial for both cell populations' cooperation. These results are promising for future clinical applications, as all components (cells, growth factors) can be prepared in an autologous way.

Platelet-released supernatant induces osteoblastic differentiation of human mesenchymal stem cells: potential role of BMP-2.

Platelet-rich preparations have recently gained popularity in maxillofacial and dental surgery, but their beneficial effect is still under debate. Furthermore, very little is known about the effect of platelet preparations at the cellular level, and the underlying mechanisms. In this study, we tested the effect of platelet-released supernatant (PRS) on human mesenchymal stem cell (MSC) differentiation towards an osteoblastic phenotype in vitro. Cultures of MSC were supplemented with PRS and typical osteoblastic markers were assessed at up to 28 days post-confluence. PRS showed an osteoinductive effect on MSC, as shown by an increased expression of typical osteoblastic marker genes such as collagen Ialpha1, bone sialoprotein II, BMP-2 and MMP-13, as well as by increased 45Ca²+ incorporation. Our results suggest that the effect of PRS on human MSC could be at least partially mediated by BMP-2. Activated autologous PRS could therefore provide an alternative to agents like recombinant bone growth factors by increasing osteoblastic differentiation of bone precursor cells at bone repair sites, although further studies are needed to fully support our observations.

In vivo biocompatibility and vascularization of biodegradable porous polyurethane scaffolds for tissue engineering.

Scaffolds for tissue engineering should be biocompatible and stimulate rapid blood vessel ingrowth. Herein, we analyzed in vivo the biocompatibility and vascularization of three novel types of biodegradable porous polyurethane scaffolds. The polyurethane scaffolds, i.e., PU-S, PU-M and PU-F, were implanted into dorsal skinfold chambers of BALB/c mice. Using intravital fluorescence microscopy we analyzed vascularization of the implants and venular leukocyte-endothelial cell interaction in the surrounding host tissue over a 14 day period. Incorporation of the scaffolds was analyzed by histology, and a WST-1 assay was performed to evaluate their cell biocompatibility in vitro. Our results indicate that none of the polyurethane scaffolds was cytotoxic. Accordingly, rolling and adherent leukocytes in venules of the dorsal skinfold chamber were found in a physiological range after scaffold implantation and did not significantly differ between the groups, indicating a good in vivo biocompatibility. However, the three scaffolds induced a weak angiogenic response with a microvessel density of only approximately 47-60 and approximately 3-10cm/cm(2) in the border and centre zones of the scaffolds at day 14 after implantation. Histology demonstrated that the scaffolds were incorporated in a granulation tissue, which exhibited only a few blood vessels and inflammatory cells. In conclusion, PU-S, PU-M and PU-F scaffolds may be used to generate tissue constructs which do not induce a strong inflammatory reaction after implantation into patients. However, the scaffolds should be further modified or conditioned in order to accelerate and improve the process of vascularization.

ADAM gene expression and regulation during human osteoclast formation.

In this study, we identified the expression and the regulation of ADAM members (a disintegrin and metalloprotease) at both gene and protein levels during human osteoclast differentiation and activity. Human peripheral blood monocytes (HPBMC) treated with M-CSF and RANKL were used as an in vitro fusion model. In parallel, we used human osteoclastoma (OCL) tumor as a source of mature osteoclasts, and human osteoblastic cells as a control representing nonfusing and non-resorbing bone cells. RT-PCR using ADAM-specific primers enabled us to identify the expression of ADAM 8, 9, 10, 15, 17, and 28 in both osteoclasts and osteoblasts. Using primers specific for each ADAM 12 isoform (L and S), we observed a strong signal for both forms (ADAM 12L and ADAM 12S) in osteoblastic cells, while only ADAM 12S was detectable in HPBMC-derived osteoclasts and osteoclastoma. Gene regulation was studied using real-time PCR analysis performed during HPBMC differentiation; this showed a progressive increase of ADAM 12 mRNA level from day 1 to 8 of the culture, while at around day 9, ADAM 12 mRNA level decreased 2-fold. We also showed that ADAM 8, ADAM 17, and ADAM 28 decreased according to the stage of HPBMC differentiation or fusion. ADAM 10 was unaltered during cell fusion. However, confocal immunolocalization showed that ADAM 10 protein re-localized from the nuclei and cytoplasm to the plasma membrane during culture and to the ruffled border in resorbing cells. The same re-localization process was observed using an ADAM 12S-specific antibody during HPBMC differentiation. Between days 12 and 14, ADAM 12 co-localized with the F-actin ring, and at day 15, a strong signal was also present in ruffled border or sealing zone area of osteoclasts. Our results describe the expression and regulation of various ADAMs in human bone cells and the selective expression of ADAM 12L in osteoblasts. Our gene regulation and protein localization studies suggest a function for ADAM 10 and ADAM 12S in the formation of osteoclasts from HPBMC and resorption activity.

In situ monitoring of cell death using Raman microspectroscopy.

We investigated the use of Raman microspectroscopy to monitor the molecular changes in human lung carcinoma epithelial cells (A549) when cell death was induced by a toxic chemical. We treated A549 cells with 100 microM Triton X-100 and carried out Raman microspectroscopy measurements in parallel with cell viability and DNA integrity assays at time points of 0, 24, 48, and 72 hours. We found that the important biochemical changes taking place during cell death, such as the degradation of proteins, DNA breakdown, and the formation of lipid vesicles, can be detected with Raman microspectroscopy. A decrease in the intensity of the O-P-O stretching Raman peak corresponding to the DNA molecule phosphate-sugar backbone at 788 cm(-1) indicated DNA disintegration, an observation which was confirmed by DNA integrity analysis. We also found a decrease in the intensity of the Raman peaks corresponding to proteins (1005 cm(-1), 1342 cm(-1)) and an increase in the concentration of lipids (1660 cm(-1), 1303 cm(-1)). These changes are the effects of the complex molecular mechanisms during the induction of cell death, such as protein cleavage due to the activation of caspases, followed by DNA fragmentation.

Spectroscopic study of human lung epithelial cells (A549) in culture: living cells versus dead cells.

The noninvasive analysis of living cells grown on 3-dimensional scaffold materials is a key point in tissue engineering. In this work we show the capability of Raman spectroscopy for use as a noninvasive method to distinguish cells at different stages of the cell cycle and living cells from dead cells. The spectral differences between cells in different stages of the cell cycle are characterized mainly by variations in DNA vibrations at 782, 788, and 1095 cm(-1). The Raman spectrum of dead human lung derived (A549 line) cells indicates the breakdown of both phosphodiester bonds and DNA bases. The most sensitive peak for identifying dead cells is the 788 cm(-1) peak corresponding to DNA Obond;Pbond;O backbone stretching. The magnitude of this peak is reduced by 80% in the spectrum of dead cells. Changes in protein peaks suggest significant conformational changes; for example, the magnitude of the 1231 cm(-1) peak assigned to random coils is reduced by 63% for dead cells. The sharp peak of phenylalanine at 1005 cm(-1) drops to half, indicating a decrease of stable proteins associated with cell death. The differences in the 1190-1385 cm(-1) spectral region also suggest a decrease in the amount of nucleic acids and proteins. Using curve fitting, we quantify these spectral differences that can be used as markers of cell death.

Function of linear and cyclic RGD-containing peptides in osteoprogenitor cells adhesion process.

Cell adhesion directly influences cell growth, differentiation and migration as well as morphogenesis, integrity and repair. The extracellular matrix (ECM) elaborated by osteoblast cells constitutes a regulator of the cell adhesion process and then of the related phenomenon. These regulatory effects of ECM are mediated through integrins and some of them are able to bind RGD sequences. The aim of this study was to determine the role of the sequence and the structure of RGD-containing peptides (linear and cyclic) as well as their role in the cell adhesion process. Cell adhesion assays onto ECM proteins coated surfaces were performed using a range of linear and cyclic RGD-containing peptides. We showed a different human osteoprogenitor cell adhesion according to the coating for ECM proteins and for RGD-peptides. Inhibition assays using peptides showed different responses depending on the coated protein. Depending on the amino-acid sequence and the structure of the peptides (cyclic linear), we observed 100% inhibition of cell adhesion onto vitronectin. These results suggest the importance of sequence, structure and conformation of the peptide, which may play a crucial function in the ligand/receptor interaction and/or in the stability of the interaction.

Complementarity between the mRNA 5' untranslated region and 18S ribosomal RNA can inhibit translation.

In eubacteria, base pairing between the 3' end of 16S rRNA and the ribosome-binding site of mRNA is required for efficient initiation of translation. An interaction between the 18S rRNA and the mRNA was also proposed for translation initiation in eukaryotes. Here, we used an antisense RNA approach in vivo to identify the regions of 18S rRNA that might interact with the mRNA 5' untranslated region (5' UTR). Various fragments covering the entire mouse 18S rRNA gene were cloned 5' of a cat reporter gene in a eukaryotic vector, and translation products were analyzed after transient expression in human cells. For the largest part of 18S rRNA, we show that the insertion of complementary fragments in the mRNA 5' UTR do not impair translation of the downstream open reading frame (ORF). When translation inhibition is observed, reduction of the size of the complementary sequence to less than 200 nt alleviates the inhibitory effect. A single fragment complementary to the 18S rRNA 3' domain retains its inhibitory potential when reduced to 100 nt. Deletion analyses show that two distinct sequences of approximately 25 nt separated by a spacer sequence of 50 nt are required for the inhibitory effect. Sucrose gradient fractionation of polysomes reveals that mRNAs containing the inhibitory sequences accumulate in the fractions with 40S ribosomal subunits, suggesting that translation is blocked due to stalling of initiation complexes. Our results support an mRNA-rRNA base pairing to explain the translation inhibition observed and suggest that this region of 18S rRNA is properly located for interacting with mRNA.

Tools for tissue engineering of mineralized oral structures.

This paper presents a short review of three groups of tools which can be or are used for the tissue engineering of mineralized oral structures: growth factor delivery systems (GFDS) and surface bioactivation with covalent bound peptides or with nanomechanically linked proteins. According to the reported personal experience of the authors, GFDS have to face the following challenging issue before being used routinely in dentistry, e.g., as a tool for reparative dentinogenesis or bone healing: adaptation of the GFDS design to the tissue where it will be implanted in order to deliver the right dose of growth factor (GF) at the right time. The bioactivation of surfaces, for example of dental implants, with covalent bound peptides or nanomechanically linked proteins represents a second innovative way to improve dental health in the future. Here we report on the experimental use of cyclic RGD peptides grafted on polymethylmethacrylate to improve osteoblast adhesion. Furthermore, we show the potential advantage of immobilizing and incorporating collagen I on titanium implant surfaces. These techniques or a combination of them will help to create improvements, for example, of dental implants in the near future. They will also help to promote bone and dentin regeneration.

Induced tissue integration of bone implants by coating with bone selective RGD-peptides in vitro and in vivo studies.

The optimal function of medical implant materials used in tissue substitution is often limited due to its healing properties. This effect is linked to reduced interactions of the implants with the surrounding tissue. Implant surfaces biologically functionalized with arginine-glycine-aspartic acid (RGD) peptides, a class of cellular adhesion factors, are described in this paper. The RGD-peptides are either bound via bovine serum albumin linking on culture plastic dishes as a model surface or via acrylic acid coupling on PMMA surface as a potential implant material. Resulting functionalized surfaces aquire the capability to bind cultured osteoblasts in high levels and show high proliferation rates in vitro. These results are observed for osteoblast cultures as well as from different species with different preparation procedures. A critical minimum distance between the bioactive portion of the RGD-peptides and the implant surface of 3.0-3.5 nm is crucial for the induction of an optimum cell binding process. In vivo animal studies in the rabbit show that newly formed bone tissue generated a direct contact with the RGD-peptide coated implants. In contrast uncoated implants are separated from newly formed bone tissue by a fibrous tissue layer thereby preventing the formation of a direct implant-bone bonding.

The product of the mammalian orthologue of the Saccharomyces cerevisiae HBS1 gene is phylogenetically related to eukaryotic release factor 3 (eRF3) but does not carry eRF3-like activity.

We describe here the cloning and sequencing of human and mouse cDNAs encoding a putative GTP binding protein. Sequence comparison shows that these cDNAs (named eRFS) are likely to represent the orthologues of the yeast Saccharomyces cerevisiae HBS1 gene and that the C-terminal domains of the encoded proteins share structural features with eukaryotic elongation factor eEF-1A and release factor 3 (eRF3) families. The phylogenetic analysis suggests that eRFS proteins and Hbs1p form a cluster of orthologous sequences branching with the eRF3 family. Nevertheless, in yeast, the human eRFS protein and Hbs1p do not complement eRF3/Sup35p thermosensitive mutation and do not interact with eRF1.