Modeling Stem/Progenitor Cell-Induced Neovascularization and\ud Oxygenation around Solid Implants

Tissue engineering constructs and other solid implants with biomedical applications, such as drug delivery devices or bioartificial organs, need oxygen (O2) to function properly. To understand better the vascular integration of such devices, we recently developed a novel model sensor containing O2-sensitive crystals, consisting of a polymeric capsule limited by a nano-porous filter. The sensor was implanted in mice with hydrogel alone (control) or hydrogel embedded with mouse CD117/c-kit+ bone marrow progenitor cells (BMPC) in order to stimulate peri-implant neovascularization. The sensor provided local partial O2 pressure (pO2) using non-invasive electron paramagnetic resonance (EPR) signal measurements. A consistently higher level of per-implant oxygenation was observed in the cell-treatment case as compared to the control over a 10-week period. In order to provide a mechanistic explanation of these experimental observations, we present in this paper a mathematical model, formulated as a system of coupled partial differential equations, that simulates peri-implant vascularization. In the control case, vascularization is considered to be the result of a Foreign Body Reaction (FBR) while in the cell-treatment case, adipogenesis in response to paracrine stimuli produced by the stem cells is assumed to induce neovascularization. The model is validated by fitting numerical predictions of local pO2 to measurements from the implanted sensor. The model is then used to investigate further the potential for using stem cell treatment to enhance the vascular integration of biomedical implants. We thus demonstrate how mathematical modeling combined with experimentation can be used to infer how vasculature develops around biomedical implants in control and stem celltreated cases

Release of VEGF from dental implant improves osteogenetic process: Preliminary in vitro tests

INTRODUCTION: During osseointegration process, the presence of an inflammatory event could negatively influence the proper osteogenetic ability of the implant surface. In order to reduce this possibility, an implementation of angiogenetic event through the release of Vascular Endothelial Growth Factor (VEGF) can be a tool as co-factor for osteoblastic differentiation. In this paper, novel dental implant surfaces enriched with VEGF have been tested. MATERIAL AND METHODS: The ability of VEGF-enriched titanium implants to improve the osteogenetic properties of Mesenchymal stem cells (MSC), also in the presence of an inflammatory environment, have been in vitro tested. Molecular biology, morphological analyses, and biochemical tests have been performed in order to confirm biological properties of these surfaces. RESULTS: Our results confirm that the presence of VEGF onto the implant surface is able not only to protect the cells from in vitro aging and from Reactive Oxygen Species (ROS) damage, but it also improves their osteogenic and endothelial differentiation, even in the presence of inflammatory cytokines. CONCLUSION: This study establishes a biologically powerful novel tool that could enhance bone repair in dental implant integration

Histomorphometric evaluation of bone regeneration induced by biodegradable scaffolds as carriers for dental pulp stem cells in a rat model of calvarial "critical size" defect

Objective: The aim of this study was to test specific stem cells that could enhance bone formation in combination with specific scaffolds. Methods: Dental Pulp Stem Cells (DPSCs) were seeded with Granular Deproteinized Bovine Bone (GDPB) or Beta-Tricalcium Phosphate (ß-TCP) in a rat model of calvarial "critical size" defect. DPSCs were isolated from permanent human teeth, obtained and characterized using specific stem cells markers (Nanog and Oct-4) by real time-PCR and immunofluorescence. Cells were differentiated for 10-15 days towards the osteoblastic phenotype with 100μM L-ascorbic acid, added every day in culture medium and 20 vol. percentage of FBS in α-MEM medium. Osteogenic commitment was evaluated with real time-PCR by measuring the expression of specific markers (osteonectin and runx2). When a sufficient cell number was obtained, DPSCs were trypsinized, washed in culture medium and seeded onto the GDPB and ß-TCP scaffold sat a density of 0.5-1×106 cells/scaffold. Two bilateral critical-size circular defects (5 mm diameter; 1 mm thickness) were created from the parietal bone of the 8 athymic T-cell deficient nude rats. One cranial defect for each rat was filled with the scaffold alone and the other defect with the scaffold seeded with stem cells. After 12 weeks post-surgery animals were euthanized and histomorphometric analysis was performed. Differences between groups were analyzed by one-way analysis of variance (ANOVA) followed by Fisher's Protected Least Significant Difference (PLSD) post-hoc test. A p-value <0.05 was considered statistically significant. Results: GDPB group presented higher percentage of lamellar bone than that of GDPB/DPSC, ß-TCP alone had lower levels as compared to ß-TCP/DPSC. The addition of stem cells significantly increased woven bone formation in both scaffold-based implants, although still higher in GDPB based implants. Conclusion: Our findings indicate that GDPB and ß-TCP used as scaffold to induce bone regeneration may benefit from adding DPSC to tissue-engineered constructs

Carboxyl-modified single-wall carbon nanotubes improve bone tissue formation in vitro and repair in an in vivo rat model.

The clinical management of bone defects caused by trauma or nonunion fractures remains a challenge in orthopedic practice due to the poor integration and biocompatibility properties of the scaffold or implant material. In the current work, the osteogenic properties of carboxyl-modified single-walled carbon nanotubes (COOH-SWCNTs) were investigated in vivo and in vitro. When human preosteoblasts and murine embryonic stem cells were cultured on coverslips sprayed with COOH-SWCNTs, accelerated osteogenic differentiation was manifested by increased expression of classical bone marker genes and an increase in the secretion of osteocalcin, in addition to prior mineralization of the extracellular matrix. These results predicated COOH-SWCNTs' use to further promote osteogenic differentiation in vivo. In contrast, both cell lines had difficulties adhering to multi-walled carbon nanotube-based scaffolds, as shown by scanning electron microscopy. While a suspension of SWCNTs caused cytotoxicity in both cell lines at levels >20 μg/mL, these levels were never achieved by release from sprayed SWCNTs, warranting the approach taken. In vivo, human allografts formed by the combination of demineralized bone matrix or cartilage particles with SWCNTs were implanted into nude rats, and ectopic bone formation was analyzed. Histological analysis of both types of implants showed high permeability and pore connectivity of the carbon nanotube-soaked implants. Numerous vascularization channels appeared in the formed tissue, additional progenitor cells were recruited, and areas of de novo ossification were found 4 weeks post-implantation. Induction of the expression of bone-related genes and the presence of secreted osteopontin protein were also confirmed by quantitative polymerase chain reaction analysis and immunofluorescence, respectively. In summary, these results are in line with prior contributions that highlight the suitability of SWCNTs as scaffolds with high bone-inducing capabilities both in vitro and in vivo, confirming them as alternatives to current bone-repair therapies

Characteristics of Alveolar Bone Marrow Cells from Patients Undergoing Dental Extractions or Dental Implant Therapy

Alveolar bone marrow stromal cells (aBMSCs) play important roles in craniofacial wound healing. To establish an easy, efficient and reliable method to harvest aBMSCs, we compared three different methods: extraction socket aspiration, osteotomy aspiration and bone core digestion. Samples of aBMSC were collected from two groups of subjects. Group 1 (dental extraction): after dental extraction, 22.5-gauge needles were used to collect 0.5-1cc marrow aspirate. Group 2 (dental implant): during implant surgeries, bone core and 0.5-1cc marrow aspirate were obtained from the osteotomy. Samples were cultured in petri dishes and attached cells were expanded. The population doubling time (PDT), surface markers, and osteogenic differentiation potential of these cells were studied. In total 12 subjects were enrolled in the study. The success rates of generating aBMSCs from extraction socket aspiration, osteotomy aspiration and bone core digestion were 42.8% (3/7), 40% (2/5) and 80% (4/5), respectively. Cells from extraction socket aspiration had the fastest proliferation rate among the three sample types, followed by bone core and osteotomy aspiration, as shown in PDTs and DNA fold changes. After isolation and expansion, all the aBMSCs expressed high levels of CD 73, CD90, and CD105, however, the expression of CD146 varied among the cells. Cells derived from bone core had the highest ALP activity after osteogenic induction, followed by cells from osteotomy aspiration, and then extraction aspiration. Taken together, bone core samples obtained during implant surgery is a more reliable source for generating aBMSCs and aBMSCs harvested from different methods may have different characteristics

Bone growth following demineralized bone matrix implantation requires angiogenesis

Angiogenesis is required for endochondral ossification during development and fracture healing; however the exact mechanisms and temporal relationship between the two processes remains unclear. In this study, we utilize an in vivo model of endochondral ossification in mice by implanting demineralized bone matrix (DBM) proximal to the femur to induce ectopic bone formation. TNP-470, a drug known to be anti-angiogenic, was used to inhibit vascularization during the time course of de novo bone formation in order to define the role of angiogenesis during the chondrogenic phase of endochondral bone formation. Day 2, day 8, and day 16 post-surgery were selected time points to represent pre-chondrogenic, chondrogenic, and bone mineralization stages, respectively. Plain x-ray and micro-CT analysis showed that inhibition of angiogenesis led to decreased mineralized tissue formation. Inhibited angiogenesis was confirmed with qRT-PCR. Most striking, however, is that while stem cells are recruited and committed to the chondrogenic lineage, subsequent chondrogenesis failed to progress based on the failure of Sox5 and Sox6 expression, which directs chondrocyte commitment. This expands the role for angiogenesis to a much earlier stage than currently thought and places the necessity of angiogenesis very early in the endochondral ossification process

The biological properties of OGI surfaces positively act on osteogenic and angiogenic commitment of mesenchymal stem cells

Osteogenesis process displays a fundamental role during dental implant osteointegration. In the present work, we studied the influence of Osteon Growth Induction (OGI) surface properties on the angiogenic and osteogenic behaviors of Mesenchymal Stem cells (MSC). MSC derived from dental pulp and HUVEC (Human Umbilical Vein Endothelial Cells) were grown in on OGI titanium surfaces, and cell proliferation and DNA synthesis were evaluated by MTT [3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide] test and DNA quantification. Gene expression has been performed in order to evaluate the presence of mRNA related to endothelial and osteogenesis markers. Moreover, morphological and biochemical analyses of osteogenesis commitments has been performed. On OGI surfaces, MSC and HUVEC are able to proliferate. Gene expression profiler confirms that MSC on OGI surfaces are able to express endothelial and osteogenic markers, and that these expression are higher compared the expression on control surfaces. In conclusion On OGI surfaces proliferation, expression and morphological analyses of angiogenesis-associated markers in MSC are promoted. This process induces an increasing on their osteogenesis commitmen

A Current Overview of Materials and Strategies for Potential Use in Maxillofacial Tissue Regeneration

Tissue regeneration is rapidly evolving to treat anomalies in the entire human body. The production of biodegradable, customizable scaffolds to achieve this clinical aim is dependent on the interdisciplinary collaboration among clinicians, bioengineers and materials scientists. While bone grafts and varying reconstructive procedures have been traditionally used for maxillofacial defects, the goal of this review is to provide insight on all materials involved in the progressing utilization of the tissue engineering approach to yield successful treatment outcomes for both hard and soft tissues. In vitro and in vivo studies that have demonstrated the restoration of bone and cartilage tissue with different scaffold material types, stem cells and growth factors show promise in regenerative treatment interventions for maxillofacial defects. The repair of the temporomandibular joint (TMJ) disc and mandibular bone were discussed extensively in the report, supported by evidence of regeneration of the same tissue types in different medical capacities. Furthermore, in addition to the thorough explanation of polymeric, ceramic, and composite scaffolds, this review includes the application of biodegradable metallic scaffolds for regeneration of hard tissue. The purpose of compiling all the relevant information in this review is to lay the foundation for future investigation in materials used in scaffold synthesis in the realm of oral and maxillofacial surgery

Performance of mesenchymal cell-scaffold constructs in human oral reconstructive surgery: a systematic review

Background: Different sources of cultured cells combined with different scaffolds (allogenic, xenogeneic, alloplastic or composite materials) have been tested extensively in vitro and in preclinical animal studies, but there have been only a few clinical trials involving humans. Aim: This study reviewed all of the English language literature published between January 1990 and December 2015 to assess the histological performance of different mesenchymal cell-scaffold constructs used for bone regeneration in human oral reconstructive procedures. Methods: An electronic search of the MEDLINE and Cochrane Central Register of Controlled Trials databases complemented by manual searching was conducted to identify studies involving histological evaluation of mesenchymal cell-scaffold constructs in human oral surgical procedures. The methodological quality of randomized controlled clinical trials and controlled clinical trials was assessed using the Cochrane Collaboration tool for assessing the risk of bias. Heterogeneity was assessed using Review Manager software. Considering the heterogeneity, the data collected were reported by descriptive methods and a meta-analysis was applied only to the articles that reported the same outcome measures. The articles were classified and described based on the material scaffolds used. Results: The search identified 1030 titles and 287 abstracts. Full-text analysis was performed for 32 articles, revealing 14 studies that fulfilled the inclusion criteria. Three randomized controlled clinical trials were identified as potentially eligible for inclusion in a meta-analysis. The studies were grouped according to the scaffold materials used: bone allograft (three studies), polyglycolic-polylactic scaffold (four studies), collagen sponge (two studies), and bovine bone matrix (five studies). The stem cells used in these studies had been sourced from the iliac crest, periosteum, dental pulp and intraoral sites. Conclusions: The very small amount of available data makes it impossible to draw any firm conclusions regarding the increase in bone formation in human oral reconstructive procedures when using graft materials engineered with autogenous stem cells

Bioactive sphene-based ceramic coatings on cpTi substrates for dental implants: An in vitro study

Titanium implant surface modifications have been widely investigated to favor the process of osseointegration. The present work aimed to evaluate the effect of sphene (CaTiSiO5) biocoating, on titanium substrates, on the in vitro osteogenic differentiation of Human Adipose-Derived Stem Cells (hADSCs). Sphene bioceramic coatings were prepared using preceramic polymers and nano-sized active fillers and deposited by spray coating. Scanning Electron Microscopy (SEM) analysis, surface roughness measurements and X-ray diffraction analysis were performed. The chemical stability of the coatings in Tris-HCl solution was investigated. In vitro studies were performed by means of proliferation test of hADSCs seeded on coated and uncoated samples after 21 days. Methyl Thiazolyl-Tetrazolium (MTT) test and immunofluorescent staining with phalloidin confirmed the in vitro biocompatibility of both substrates. In vitro osteogenic differentiation of the cells was evaluated using Alizarin Red S staining and quantification assay and real-time PCR (Polymerase Chain Reaction). When hADSCs were cultured in the presence of Osteogenic Differentiation Medium, a significantly higher accumulation of calcium deposits onto the sphene-coated surfaces than on uncoated controls was detected. Osteogenic differentiation on both samples was confirmed by PCR. The proposed coating seems to be promising for dental and orthopedic implants, in terms of composition and deposition technology