Improved implant osseointegration of a nanostructured titanium surface via mediation of macrophage polarization

The use of endosseous implanted materials is often limited by undesirable effects that may be due to macrophage-related inflammation. The purpose of this study was to fabricate a nanostructured surface on a titanium implant to regulate the macrophage inflammatory response and improve the performance of the implant. Anodization at 5 and 20 V as well as UV irradiation were used to generate hydrophilic, nanostructured TiO₂ surfaces (denoted as NT5 and NT20, respectively). Their surface characteristics and in vivo osseointegration as well as the inflammatory response they elicit were analyzed. In addition, the behavior of macrophages in vitro was evaluated. Although the in vitro osteogenic activity on the two surfaces was similar, the NT5 surface was associated with more bone formation, less inflammation, and a reduced CD68⁺ macrophage distribution in vivo compared to the NT20 and polished Ti surfaces. Consistently, further experiments revealed that the NT5 surface induced healing-associated M2 polarization in vitro and in vivo. By contrast, the NT20 surface promoted the pro-inflammatory M1 polarization, which could further impair bone regeneration. The results demonstrate the dominant role of macrophage-related inflammation in bone healing around implants and that surface nanotopography can be designed to have an immune-regulating effect in support of the success of implants.     

In vivo stimulation of bone formation by aluminum and oxygen plasma surface-modified magnesium implants

A newly developed magnesium implant is used to stimulate bone formation in vivo. The magnesium implant after undergoing dual aluminum and oxygen plasma implantation is able to suppress rapid corrosion, leaching of magnesium ions, as well as hydrogen gas release from the biodegradable alloy in simulated body fluid (SBF). No released aluminum is detected from the SBF extract and enhanced corrosion resistance properties are confirmed by electrochemical tests. In vitro studies reveal enhanced growth of GFP mouse osteoblasts on the aluminum oxide coated sample, but not on the untreated sample. In addition to that a small amount (50 ppm) of magnesium ions can enhance osteogenic differentiation as reported previously, our present data show a low concentration of hydrogen can give rise to the same effect. To compare the bone volume change between the plasma-treated magnesium implant and untreated control, micro-computed tomography is performed and the plasma-treated implant is found to induce significant new bone formation adjacent to the implant from day 1 until the end of the animal study. On the contrary, bone loss is observed during the first week post-operation from the untreated magnesium sample. Owing to the protection offered by the Al₂O₃ layer, the plasma-treated implant degrades more slowly and the small amount of released magnesium ions stimulate new bone formation locally as revealed by histological analyses. Scanning electron microscopy discloses that the Al₂O₃ layer at the bone-implant interface is still present two months after implantation. In addition, no inflammation or tissue necrosis is observed from both treated and untreated implants. These promising results suggest that the plasma-treated magnesium implant can stimulate bone formation in vivo in a minimal invasive way and without causing post-operative complications.     

Optical imaging of fibrin deposition to elucidate participation of mast cells in foreign body responses

Mast cell activation has been shown to be an initiator and a key determinant of foreign body reactions. However, there is no non-invasive method that can quantify the degree of implant-associated mast cell activation. Taking advantage of the fact that fibrin deposition is a hallmark of mast cell activation around biomaterial implants, a near infrared probe was fabricated to have high affinity to fibrin. Subsequent in vitro testing confirmed that this probe has high affinity to fibrin. Using a subcutaneous particle implantation model, we found significant accumulation of fibrin-affinity probes at the implant sites as early as 15 min following particle implantation. The accumulation of fibrin-affinity probes at the implantation sites could also be substantially reduced if anti-coagulant – heparin was administered at the implant sites. Further studies have shown that subcutaneous administration of mast cell activator – compound 48/80 – prompted the accumulation of fibrin-affinity probes. However, implant-associated fibrin-affinity probe accumulation was substantially reduced in mice with mast cell deficiency. The results show that our fibrin-affinity probes may serve as a powerful tool to monitor and measure the extent of biomaterial-mediated fibrin deposition and mast cell activation in vivo.     

Bone regeneration around N-acetyl cysteine-loaded nanotube titanium dental implant in rat mandible

New strategies involving drugs loading onto implant surfaces are required to enhance osseointegration and shorten healing time after implantation. In this study, we examined the feasibility of N-acetyl cysteine (NAC)-loaded nanotube titanium (NLN-Ti) implants as a potential drug delivery system. To determine the effect of NLN-Ti in in vitro and in vivo, viability and ROS formation was assessed and enzyme-linked immunosorbant assay (ELISA), Western blot, micro-computed tomography (μ-CT), hematoxylin and eoxin (H&E) staining and immunohistochemical (IHC) analysis were done. In vitro, cell viability was increased and inflammatory responses and reduced oxidative stress-related defense were decreased with MC 3T3-E1 cells exposed to a sustained release of NAC from NLN-Ti implants. Following NLN-Ti implant installation, μ-CT revealed an increase of newly formed bone volume and bone mineral density in the mandibles of Sprague Dawley rats. Relatively well formed new bone was demonstrated in close contact to the NLN-Ti implant surface by H&E staining. IHC revealed significantly higher expression of bone morphogenetic protein-2, -7 and heme oxygenase-1, and reduced expression of receptor activator of nuclear factor-kappa B ligand. The data indicate that NLN-Ti implants enhance osseointegration and highlight the value of the small animal model in assessing diverse biological responses to dental implants.     

Vascularization and bone regeneration in a critical sized defect using 2-N,6-O-sulfated chitosan nanoparticles incorporating BMP-2

An ideal bone tissue engineering graft should have both excellent pro-osteogenesis and pro-angiogenesis to rapidly realize the bone regeneration in vivo. To meet this goal, 2-N,6-O-sulfated chitosan (26SCS) based nanoparticle (S-NP) was successfully developed and showed a dose-dependent enhancement on angiogenesis in vitro. For the repair of a critical sized defect in rabbit radius, we developed BMP-2 loaded S-NP (BMP-2/S-NP) with protein loading efficiency of 1.4 ± 0.2% and fabricated a gelatin sponge (G) based implant loaded with BMP-2/S-NP (BMP-2/S-NP/G). This implant exerted a delivery of BMP-2 with an initial burst release of 15.3 ± 4.1% in first 24 h and a gradual release for 21 days to 77.8 ± 3.6%. The in vitro ALP assay revealed that the activity of released BMP-2 from BMP-2/S-NP/G was maintained after 3-d and 7-d delivery and further enhanced after 14-d delivery compared with the original BMP-2. Furthermore, the in vivo effects of BMP-2/S-NP/G on the bone regeneration and vessel formation in the critical sized defect (18 mm) of rabbit radius were investigated by synchrotron radiation-based micro-computed tomography (SRμCT) imaging, three dimensional micro-computed tomographic (μCT) imaging, histological analysis, immunohistochemistry and biomechanical measurement. Based on the results, both peripheral vessel and new vessel formation were significantly increased by the BMP-2/S-NP/G treatment, along with the bridged defects at as early as 2 weeks, the healed defects at 8 weeks and the reunion of bone marrow cavity at 12 weeks. The results indicated that both controlled release of active BMP-2 and favorable vascularization at the defect site contributed by BMP-2/S-NP/G played a crucial role in accelerating and promoting bone augmentation. This study suggests that BMP-2/S-NP/G demonstrates promise for vascularization and bone regeneration in clinical case of large defect.     

Zn/Ag micro-galvanic couples formed on titanium and osseointegration effects in the presence of S. aureus

Titanium implants possessing simultaneous osseointegration and antibacterial ability are desirable. In this work, three types of Zn/Ag micro-galvanic couples are fabricated on titanium by plasma immersion ion implantation to investigate the osseointegration and antibacterial effects as well as the involved mechanisms. The in vitro findings disclose enhanced proliferation, osteogenic differentiation, and gene expressions of the rat bone mesenchymal stem cells (rBMSCs), as well as good antibacterial ability on all three micro-galvanic couples. Excellent antimicrobial ability is also observed in vivo and the micro-CT and histological results reveal notable osseointegration in vivo despite the presence of bacteria. The Zn/Ag micro-galvanic couple formed on Zn/Ag dual-ion co-implanted titanium shows the best osseointegration as well as good antibacterial properties in vivo obtained from a rabbit tibia model. The difference among the three Zn/Ag micro-galvanic couples can be ascribed to the contact between the Ag NPs and Zn film, which affects the corrosion process. Our results indicate that the biological behavior can be controlled by the corrosion process of the Zn/Ag micro-galvanic couples.     

Osseointegration of machined,injection moulded and oxygen plasma modified PEEK implants in a sheep model

Machined and injection moulded polyetheretherketone (PEEK) implants with and without an oxygen plasma modification were prepared and implanted in sheep cancellous and cortical bone. After 4, 12 and 26 weeks, osseointegration was evaluated through mechanical push-out tests and histomorphometry. In the cancellous bone, push-out force increased with time, a trend toward higher force was observed for machined compared to moulded, and oxygen plasma modified compared to unmodified. On-going remodelling of the bone was detected in the periphery of the implants at 4 weeks. Minimal or no inflammation was observed with all the implants at all locations and time-points. Bone-implant contact (BIC) was quantified at all-time points and locations for all the four PEEK implant surfaces. The BIC values ranged from 15 to 75% with an average of 29 ± 13% in the cancellous bone and 25–65% with an average of 50 ± 12% in the cortical bone. In the cortical bone the BIC increased significantly from 4 to 26 weeks. This in vivo study has identified that surface topography of PEEK implants influences osseointegration. In addition, oxygen plasma has the potential to increase bone-implant interface stability. This study provides a unique reference for further modifications and in vivo assessment of PEEK implants.     

The effect of dose on rhBMP-2 signaling,delivered via collagen sponge,on osteoclast activation and in vivo bone resorption

While recombinant human bone morphogenetic protein (rhBMP)-2-based bone therapy presents potential osteoinductivity, it also leads concern due to transient osteoclast activation during early healing periods, ultimately limiting its clinical use. Therefore, we investigated in vivo and in vitro rhBMP-2 signaling which mediates early bone resorbing effect, depending on the dose, and attempted to inhibit this resorption phenomenon using NFAT inhibitor as a target molecule. High-dose of rhBMP-2 (20 μg/defect) enhanced osteoclast activation and the expression of bone resorption markers, compared to low dose (5 μg/defect) at one week after surgery in collagen sponge-delivered rat calvarial defect models. Interestingly, this trend was also observed in the expression of bone formation markers. In particular, rhBMP-2 upregulated RANKL expression, while it downregulated osteoprotegerin (OPG) expression, resulting in a dose-dependent increase in the ratio of RANKL to OPG. NFAT inhibitor (150 μm) treatment in vivo suppressed the high-dose effect of rhBMP-2 on both resorption and formation. In vitro results of rhBMP-2 signaling and NFAT inhibitor effects in rat mesenchymal stem cells showed similar trends as in vivo results. Microcomputer tomography-based evaluation at 4 weeks showed that combined treatment of NFAT inhibitor with 20 μg rhBMP-2 in vivo increased bone volume (BV) more than 20 μg rhBMP-2 alone, which showed little difference in BV compared to 5 μg of rhBMP-2. These results demonstrated that rhBMP-2 implantation concurrently signalized into enhanced osteoclastogenesis and osteoblastogenesis in vivo, dose-dependently. Ratio of RANKL/OPG might be an index for early bone resorbing activity of implanted rhBMP-2. A local cocktail treatment of NFAT inhibitor and high-dose rhBMP-2 might be an alternative to overcome early bone resorbing effects, thereby accelerating bone formation.     

In vivo real-time visualization of tissue blood flow and angiogenesis using Ag2S quantum dots in the NIR-II window

Improving the tissue penetration depth and spatial resolution of fluorescence-based optical nanoprobes remains a grand challenge for their practical applications in in vivo imaging, due to the scattering and absorption and endogenous autofluorescence of living tissues. Here, we present that Ag₂S quantum dots (QDs), containing no toxic ions, exhibiting long circulation time and high stability, act as a new kind of fluorescent probes in the second near-infrared window (NIR-II, 1000–1350 nm) which enable in vivo monitoring of lymphatic drainage and vascular networks with deep tissue penetration and high spatial and temporal resolution. In addition, NIR-II fluorescence imaging with Ag₂S QDs provide ultrahigh spatial resolution (∼40 μm) that permits us to track angiogenesis mediated by a tiny tumor (2–3 mm in diameter) in vivo. Our results indicate that Ag₂S QDs are promising NIR-II fluorescent nanoprobes that could be useful in surgical treatments such as sentinel lymph node (SLN) dissection as well in assessment of blood supply in tissues and organs and screening of anti-angiogenic drugs.     

Histological analysis of cells and matrix mineralization of new bone tissue induced in rabbit femur bones by Mg–Zr based biodegradable implants

The biological efficacy of bone inducing implant materials in situ can be assessed effectively by performing histological analysis. We studied the peri-implant bone regeneration around two types of biodegradable magnesium–zirconium alloys, Mg–5Zr and Mg–Zr–2Sr, using histological, histochemical and immunohistochemical methods in the femur of New Zealand White strain rabbits. Our study includes three animal groups: (a) Mg–5Zr, (b) Mg–Zr–2Sr and (c) control. In each group three animals were used and in groups ‘a’ and ‘b’ the respective alloys were implanted in cavities made at the distal ends of the femur; control animals were left without implants to observe natural bone healing. Qualitative assessment of the cellularity and matrix mineralization events of the newly formed bone tissue was done at three months after implantation by histological methods in methyl methacrylate embedded tissue without decalcifying the bone. Quantitative mineral content and density of the new bone (NB) were evaluated by the statistical analysis of dual energy X-ray absorptiometry (DXA) data obtained from three animals in each experimental group. Based on our analysis we conclude that Mg–Zr–2Sr alloy showed better osseointegration of the newly formed bone with the implant surface. Our methodology of studying peri-implant osteoinduction of degradable implants using low temperature methyl methacrylate embedding resin can be useful as a general method for determining the bio-efficacy of implant materials.     

Modulation of the foreign body response to implanted sensor models through device-based delivery of the tyrosine kinase inhibitor,masitinib

The host foreign body response (FBR) adversely effects the performance of numerous implanted biomaterials especially biosensors, including clinically popular glucose-monitoring sensors. Reactive formation of a fibrous capsule around implanted sensors hinders the transport of essential analytes to the sensor from the surrounding tissue, resulting in loss of glucose response sensitivity and eventual sensor failure. Several strategies have sought to mitigate the foreign body response's effects on CGM sensors through the use of local delivery of pharmaceuticals and biomolecules with limited success. This study describes release of a tyrosine kinase inhibitor – masitinib – from the sensor implant to target tissue resident mast cells as key mediators of the FBR. Model implants are coated with a composite polymer hydrophilic matrix that rapidly dissolves upon tissue implantation to deposit slower-degrading polymer microparticles containing masitinib. Matrix dissolution limits coating interference with sensor function while establishing a local controlled-release delivery depot formulation to alter implant tissue pharmacology and addressing the FBR. Drug efficacy was evaluated in a murine subcutaneous pocket implant model. Drug release extends to more than 30 days in vitro. The resulting FBR in vivo, evaluated by implant capsule thickness and inflammatory cell densities at 14, 21, and 28 days, displays statistically significant reduction in capsule thickness around masitinib-releasing implant sites compared to control implant sites.     

In vitro and in vivo studies on the degradation of high-purity Mg (99.99wt.%) screw with femoral intracondylar fractured rabbit model

High-purity magnesium (HP Mg) takes advantage in no alloying toxic elements and slower degradation rate in lack of second phases and micro-galvanic corrosion. In this study, as rolled HP Mg was fabricated into screws and went through in vitro immersion tests, cytotoxicity test and bioactive analysis. The HP Mg screws performed uniform corrosion behavior in vitro, and its extraction promoted cell viability, bone alkaline phosphatase (ALP) activity, and mRNA expression of osteogenic differentiation related gene, i.e. ALP, osteopontin (OPN) and RUNX2 of human bone marrow mesenchymal stem cells (hBMSCs).Then HP Mg screws were implanted in vivo as load-bearing implant to fix bone fracture and subsequently gross observation, range of motion (ROM), X-ray scanning, qualitative micro-computed tomography (μCT) analysis, histological analysis, bending-force test and SEM morphology of retrieved screws were performed respectively at 4, 8, 16 and 24 weeks. As a result, the retrieved HP Mg screws in fixation of rabbit femoral intracondylar fracture showed uniform degradation morphology and enough bending force. However, part of PLLA screws was broken in bolt, although its screw thread was still intact. Good osseointegration was revealed surrounding HP Mg screws and increased bone volume and bone mineral density were detected at fracture gap, indicating the rigid fixation and enhanced fracture healing process provided by HP Mg screws. Consequently, the HP Mg showed great potential as internal fixation devices in intra-articular fracture operation.     

Protein composition alters in vivo resorption of PEG-based hydrogels as monitored by contrast-enhanced MRI

We report on the use of magnetic resonance imaging (MRI)-based non-invasive monitoring to document the role of protein adjuvants in hydrogel implant integration in vivo. Polyethylene glycol (PEG) hydrogels were formed with different protein constituents, including albumin, fibrinogen and gelatin. The hydrogels were designed to exhibit similar material properties, including modulus, swelling and hydrolytic degradation kinetics. The in vivo resorption properties of these PEG-based hydrogels, which contained a tethered gadolinium contrast agent, were characterized by MRI and histology, and compared to their in vitro characteristics. MRI data revealed that PEG–Albumin implants remained completely intact throughout the experiments, PEG–Fibrinogen implants lost about 10% of their volume and PEG–Gelatin implants underwent prominent swelling and returned to their initial volume by day 25. Fully synthetic PEG–diacrylate (PEG–DA) control hydrogels lost about half of their volume after 25 days in vivo. Transverse MRI cross-sections of the implants revealed distinct mechanisms of the hydrogel's biodegradation: PEG–Fibrinogen and PEG–Albumin underwent surface erosion, whereas PEG–Gelatin and PEG–DA hydrogels mainly underwent bulk degradation. Histological findings substantiated the MRI data and demonstrated significant cellular response towards PEG–DA and PEG–Gelatin scaffolds with relatively low reaction towards PEG–Fibrinogen and PEG–Albumin hydrogels. These findings demonstrate that PEG–protein hydrogels can degrade via a different mechanism than PEG hydrogels, and that this difference can be linked to a reduced foreign body response.     

A functional biphasic biomaterial homing mesenchymal stem cells for in vivo cartilage regeneration

Cartilage regeneration after trauma is still a great challenge for clinicians and researchers due to many reasons, such as joint load-bearing, synovial movement and the paucity of endogenous repair cells. To overcome these limitations, we constructed a functional biomaterial using a biphasic scaffold platform and a bone-derived mesenchymal stem cells (BMSCs)-specific affinity peptide. The biphasic scaffold platform retains more cells homogeneously within the sol–gel transition of chitosan and provides sufficient solid matrix strength. This biphasic scaffold platform is functionalized with an affinity peptide targeting a cell source of interest, BMSCs. The presence of conjugated peptide gives this system a biological functionality towards BMSC-specific homing both in vitro and in vivo. The functional biomaterial can stimulate stem cell proliferation and chondrogenic differentiation during in vitro culture. Six months after in vivo implantation, compared with routine surgery or control scaffolds, the functional biomaterials induced superior cartilage repair without complications, as indicated by histological observations, magnetic resonance imaging and biomechanical properties. Beyond cartilage repair, this functional biphasic scaffold may provide a biomaterial framework for one-step tissue engineering strategy by homing endogenous cells to stimulate tissue regeneration.     

Circumferentially aligned fibers guided functional neoartery regeneration in vivo

An ideal vascular graft should have the ability to guide the regeneration of neovessels with structure and function similar to those of the native blood vessels. Regeneration of vascular smooth muscle cells (VSMCs) with circumferential orientation within the grafts is crucial for functional vascular reconstruction in vivo. To date, designing and fabricating a vascular graft with well-defined geometric cues to facilitate simultaneously VSMCs infiltration and their circumferential alignment remains a great challenge and scarcely reported in vivo. Thus, we have designed a bi-layered vascular graft, of which the internal layer is composed of circumferentially aligned microfibers prepared by wet-spinning and an external layer composed of random nanofibers prepared by electrospinning. While the internal circumferentially aligned microfibers provide topographic guidance for in vivo regeneration of circumferentially aligned VSMCs, the external random nanofibers can offer enhanced mechanical property and prevent bleeding during and after graft implantation. VSMCs infiltration and alignment within the scaffold was then evaluated in vitro and in vivo. Our results demonstrated that the circumferentially oriented VSMCs and longitudinally aligned ECs were successfully regenerated in vivo after the bi-layered vascular grafts were implanted in rat abdominal aorta. No formation of thrombosis or intimal hyperplasia was observed up to 3 month post implantation. Further, the regenerated neoartery exhibited contraction and relaxation property in response to vasoactive agents. This new strategy may bring cell-free small diameter vascular grafts closer to clinical application.     

Visible-light-excited and europium-emissive nanoparticles for highly-luminescent bioimaging in vivo

Europium(III)-based material showing special milliseconds photoluminescence lifetime has been considered as an ideal time-gated luminescence probe for bioimaging, but is still limited in application in luminescent small-animal bioimaging in vivo. Here, a water-soluble, stable, highly-luminescent nanosystem, Ir–Eu–MSN (MSN = mesoporous silica nanoparticles, Ir–Eu = [Ir(dfppy)₂(pic–OH)]₃Eu·2H₂O, dfppy = 2-(2,4-difluorophenyl)pyridine, pic–OH = 3-hydroxy-2-carboxypyridine), was developed by an in situ coordination reaction to form an insoluble dinuclear iridium(III) complex-sensitized-europium(III) emissive complex within mesoporous silica nanoparticles (MSNs) which had high loading efficiency. Compared with the usual approach of physical adsorption, this in-situ reaction strategy provided 20-fold the loading efficiency (43.2%) of the insoluble Ir–Eu complex in MSNs. These nanoparticles in solid state showed bright red luminescence with high quantum yield of 55.2%, and the excitation window extended up to 470 nm. These Ir–Eu–MSN nanoparticles were used for luminescence imaging in living cells under excitation at 458 nm with confocal microscopy, which was confirmed by flow cytometry. Furthermore, the Ir–Eu–MSN nanoparticles were successfully applied into high-contrast luminescent lymphatic imaging in vivo under low power density excitation of 5 mW cm⁻². This synthetic method provides a universal strategy of combining hydrophobic complexes with hydrophilic MSNs for in vivo bioimaging.     

Biocompatibility and inflammatory response in vitro and in vivo to gelatin-based biomaterials with tailorable elastic properties

Hydrogels prepared from gelatin and lysine diisocyanate ethyl ester provide tailorable elastic properties and degradation behavior. Their interaction with human aortic endothelial cells (HAEC) as well as human macrophages (M?) and granulocytes (G?) were explored. The experiments revealed a good biocompatibility, appropriate cell adhesion, and cell infiltration. Direct contact to hydrogels, but not contact to hydrolytic or enzymatic hydrogel degradation products, resulted in enhanced cyclooxygenase-2 (COX-2) expression in all cell types, indicating a weak inflammatory activation in vitro. Only M? altered their cytokine secretion profile after direct hydrogel contact, indicating a comparably pronounced inflammatory activation. On the other hand, in HAEC the expression of tight junction proteins, as well as cytokine and matrix metalloproteinase secretion were not influenced by the hydrogels, suggesting a maintained endothelial cell function. This was in line with the finding that in HAEC increased thrombomodulin synthesis but no thrombomodulin membrane shedding occurred. First in vivo data obtained after subcutaneous implantation of the materials in immunocompetent mice revealed good integration of implants in the surrounding tissue, no progredient fibrous capsule formation, and no inflammatory tissue reaction in vivo. Overall, the study demonstrates the potential of gelatin-based hydrogels for temporal replacement and functional regeneration of damaged soft tissue.     

Mesenchymal stem cell characteristics of dental pulp and periodontal ligament stem cells after in vivo transplantation

Mesenchymal stem cells (MSCs) isolated from human postnatal dental pulp and periodontal ligament (PDL) tissues can give rise to multilineage differentiation in vitro and generate related dental tissues in vivo. However, the cell properties of human dental pulp stem cells (DPSCs) and PDL stem cells (PDLSCs) after in vivo implantation remain largely unidentified. In this study, cells were re-isolated from in vivo-generated dental pulp-like and PDL-like tissues (termed re-DPCs and re-PDLCs, respectively) as a result of ectopic transplantation of human DPSC and PDLSC sheets. The cell characteristics in terms of colony-forming ability, cell surface antigens and multi-differentiation potentials were all evaluated before and after implantation. It was found that re-DPCs and re-PDLCs were of human and mesenchymal origin and positive for MSC markers such as STRO-1, CD146, CD29, CD90 and CD105; and, to some extent, re-DPCs could maintain their colony forming abilities. Moreover, both cell types were able to form mineral deposits and differentiate into adipocytes and chondrocytes; however, quantitative analysis and related gene expression determination showed that the osteo-/chondro-differentiation capabilities of re-DPCs and re-PDLCs were significantly reduced compared to those of DPSCs and PDLSCs, respectively (P < 0.05); re-PDLCs showed a greater reduction potential than re-DPCs. We conclude that DPSCs and PDLSCs may maintain their MSC characteristics after in vivo implantation and, compared to PDLSCs, DPSCs appear much more stable under in vivo conditions. These findings provide additional cellular and molecular evidence that supports expanding the use of dental tissue-derived stem cells in cell therapy and tissue engineering.     

Controlling stem cell-mediated bone regeneration through tailored mechanical properties of collagen scaffolds

Mechanical properties of the extracellular matrix (ECM) play an essential role in cell fate determination. To study the role of mechanical properties of ECM in stem cell-mediated bone regeneration, we used a 3D in vivo ossicle model that recapitulates endochondral bone formation. Three-dimensional gelatin scaffolds with distinct stiffness were developed using 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) mediated zero-length crosslinking. The mechanical strength of the scaffolds was significantly increased by EDC treatment, while the microstructure of the scaffold was preserved. Cell behavior on the scaffolds with different mechanical properties was evaluated in vitro and in vivo. EDC-treated scaffolds promoted early chondrogenic differentiation, while it promoted both chondrogenic and osteogenic differentiation at later time points. Both micro-computed tomography and histologic data demonstrated that EDC-treatment significantly increased trabecular bone formation by transplanted cells transduced with AdBMP. Moreover, significantly increased chondrogenesis was observed in the EDC-treated scaffolds. Based on both in vitro and in vivo data, we conclude that the high mechanical strength of 3D scaffolds promoted stem cell mediated bone regeneration by promoting endochondral ossification. These data suggest a new method for harnessing stem cells for bone regeneration in vivo by tailoring the mechanical properties of 3D scaffolds.     

Calvarial defect healing by recruitment of autogenous osteogenic stem cells using locally applied simvastatin

Local statins implant has been shown to promote bone healing, the underlying mechanisms are unclear. The purpose of this study was to test the effect of local simvastatin implant on bone defect healing; to evaluate the mobilization, migration, and homing of bone marrow-derived mesenchymal stem cells (BMSCs) and endothelial progenitor cells (EPCs) induced by simvastatin. We found that local simvastatin implant increased bone formation by 51.8% (week 6) and 64.8% (week 12) compared with polyglycolic acid controls (P < 0.01), as verified by X-ray, CT, and histology. Simvastatin increased migration capacity of BMSCs and EPCs in vitro (P < 0.05). Local simvastatin implant increased mobilization of EPCs to the peripheral blood by 127% revealed by FACS analysis (P < 0.01), and increased osteogenic BMSCs to the peripheral blood dramatically revealed by Alizarin Red-S staining for mineralized nodules formation. Pre-transplanted GFP-transfected BMSCs as a tracing cell and bioluminescence imaging revealed that local simvastatin implant recruited GFP-labeled BMSC. Also, local simvastatin implant induced the HIF-1α and BMP-2 expression. In conclusion, local simvastatin implantation promotes bone defect healing, where the underlying mechanism appears to involve the higher expression of HIF-1α and BMP-2, thus recruit autogenous osteogenic and angiogenetic stem cells to the bone defect area implanted with simvastatin.