Assessment of bone regeneration of a tissue-engineered bone complex using human dental pulp stem cells/poly(ε-caprolactone)-biphasic calcium phosphate scaffold constructs in rabbit calvarial defects

The objective of the present study was to investigate the effect of a fabricated combination of poly-?-caprolactone (PCL)–biphasic calcium phosphate (BCP) with the modified melt stretching and multilayer deposition (mMSMD) technique on human dental pulp stem cell (hDPSC) differentiation to be osteogenic like cells for bone regeneration of calvarial defects in rabbit models. hDPSCs extracted from human third molars were seeded onto mMSMD PCL-BCP scaffolds and the osteogenic gene expression was tested prior to implantation in vivo. Two standardized 11?mm in diameter circular calvarial defects were created in 18 adult male New Zealand white rabbits. The rabbits were divided into 4 groups: (1) hDPSCs seeded in mMSMD PCL-BCP scaffolds; (2) mMSMD PCL-BCP scaffolds alone, (3) empty defects and (4) autogenous bone (n?=?3 site/time point/groups). After two, four and eight weeks after the operation, the specimens were harvested for micro-CT including histological and histomorphometric analysis. The explicit results presented an interesting view of the bioengineered constructs of hDPSCs in PCL-BCP scaffolds that increased the newly formed bone compared to the empty defect and scaffold alone groups. The results demonstrated that hDPSCs combined with mMSMD PCL-BCP scaffolds may be an augmentation material for bony defect.     

LvBMP-2 gene-modified BMSCs combined with calcium phosphate cement scaffolds for the repair of calvarial defects in rats

The study aims to evaluate the effect of bone marrow stromal cells (BMSCs) expressing bone morphogenic protein-2 (BMP-2) mediated by lentiviral (Lv) gene transduction combined with calcium phosphate cement (CPC) scaffolds for the repair of critical size calvarial defects in rats. BMSCs derived from Fisher 344 rats were transduced with LvBMP-2 or lentivirus encoding enhanced green fluorescent protein (LvEGFP) in vitro. Obvious osteogenic differentiation of BMSCs in the LvBMP-2 group was demonstrated by alkaline phosphatase staining and alizarin red staining. Enzyme-linked immunosorbent assay results show that LvBMP-2 gene expression in vitro can last for at least 8 weeks. Gene-transduced or untransduced BMSCs were seeded onto CPC scaffolds to repair rat calvarial defects with a diameter of 5 mm. Scanning electron microscope analysis indicated that porous CPC scaffolds facilitated initial adhesion and spreading of BMSCs onto its surface. Calvarial defects were successfully repaired with LvBMP-2-transduced BMSCs/CPC constructs 8 weeks postoperatively. The percentage of new bone formation in the LvBMP-2 group was significantly higher than in other control groups. Lentiviral mediated BMP-2 gene therapy together with CPC scaffolds can be used successfully in calvarial repair and bone regeneration.     

Injectable silk/hydroxyapatite nanocomposite hydrogels with vascularization capacity for bone regeneration

Localized and sustained osteogenic-angiogenic stimulation to bone defects is critical for effective bone repair.Here,desferrioxamine(DFO)was loaded on silk fibroin nanofibers and blended with hydroxyapatite nanorods(HA),forming injectable DFO-loaded silk fibroin-HA nanocomposite hydrogels.The composite hydrogels remained homogeneous distribution of HA with high ratio(60%)and also higher stiffness than that of pure silk fibroin nanofiber hydrogels,which provided stable osteogenic stimulation niches for tissue regeneration.Without the scarify of injectability,the hydrogels achieved slow delivery of DFO for above 60 days,resulting in suitable angiogenesis in vitro and in vivo and better osteogenesis than DFO-free systems.Compared to previous injectable silk fibroin-HA hydrogels,the introduction of vascularization capacity further stimulated the osteogenic differentiation of stem cells and accelerated new bone formation.Quicker and better bone healing were detected at defect sites after the injection of DFO-loaded nanocomposite hydrogels,indicating the effective synergistic effect of osteogenic and angiogenic cues.This work provides a simple and effective strategy of introducing angiogenic cues to bone matrices.We believe that the injectable nanocomposite hydrogels are suitable for the regeneration of bone tissues.     

Analysis of OPLA scaffolds for bone engineering constructs using human jaw periosteal cells

For bone regeneration constructs using human jaw periosteal cells (JPC) the extent of osteoinductive ability of different three-dimensional scaffolds is not yet established. We analyzed open-cell polylactic acid (OPLA) scaffolds for their suitability as bone engineering constructs using human JPC. Cell adhesion and spreading was visualized on the surface of scaffolds by scanning electron microscopy. JPC proliferation within OPLA scaffolds was compared with proliferation within collagen and calcium phosphate scaffolds. We found a significant increase of proliferation rates in OPLA scaffolds versus Coll/CaP scaffolds at three time points. Live-measurements of oxygen consumption within the cell-seeded scaffolds indicate that the in vitro culturing time should not exceed 12–15 days. OPLA scaffolds, which were turned out to be the most beneficial for JPC growth, were chosen for osteogenic differentiation experiments with or without BMP-2. Gene expression analyses demonstrated induction of several osteogenic genes (alkaline phosphatase, osterix, Runx-2 and insulin-like growth factor) within the 3D-scaffolds after 12 days of in vitro culturing. Element analysis by EDX spectrometry of arising nodules during osteogenesis demonstrated that JPC growing within OPLA scaffolds are able to form CaP particles. We conclude that OPLA scaffolds provide a promising environment for bone substitutes using human JPC.     

In vivo study of a bioactive nanoparticle-gelatin composite scaffold for bone defect repair in rabbits

The purpose is to study the in vivo bioactivity of this scaffold and verify its ability to simulate the characteristics of cancellous bone. Twenty-four adult New Zealand white rabbits were divided into three groups. Bone defects above the femoral condylar of both sides were created. A newly designed bioactive nanoparticle–gelatin composite scaffold was implanted to the experimental side, while the control side was left without implantation. The repair of bone defect was monitored by X-ray examination, gross observation, Micro-CT examination and histological observation of the area of bone defect 4, 8 and 12 weeks after surgery. There was void of new bone tissue in medullary cavity in the bone defect area of the control side. In the experimental side, the composite scaffold displayed excellent biodegradability, bioactivity and cyto-compatibility. With the time laps, new bone tissue grew from the edge to center as revealed by both Micro-CT image and staining biopsy, which complies with the “creeping substitution” process. The mechanical properties of the newly designed bioactive nanoparticle–gelatin composite scaffold and the 3-D structure of new bone tissue are comparable to the surrounding cancellous bones. This newly developed bioactive nanoparticle–gelatin composite scaffold possesses good biocompatibility and in vivo osteogenic capability for bone defect repair. It may be a promising artificial bone grafts.     

Triphasic ceramic coated hydroxyapatite as a niche for goat stem cell-derived osteoblasts for bone regeneration and repair

Current treatment strategies for the repair or replacement of bone use synthetic implants with stem cells and their progeny--a new approach to address unmet medical needs. This study has evaluated the effect of a silica-coated bioactive ceramic, namely HASi in comparison to hydroxyapatite (HA) on the adhesion, proliferation and osteogenic differentiation of goat bone marrow-derived mesenchymal stem cells in vitro in a prolonged culture of 28 days. The cellular activities were significantly enhanced on HASi signifying the role of silica to stimulate osteoblast cells. The fabrication of such a 'cell-ceramic construct using autologous MSCs' is aimed for the transplantation to a large bone defect site in the goat femur model which still remains a formidable challenge in Orthopedic surgery.     

Three-dimensionally plotted MBG/PHBHHx composite scaffold for antitubercular drug delivery and tissue regeneration

A suitable drug-loaded scaffold that can postoperatively release an antituberculosis drug efficiently in a lesion area and help repair a bone defect is very important in the clinical treatment of bone tuberculosis (TB). In this study, a composite drug-loaded cylindrical scaffold was prepared by using three-dimensional printing technology in combination with the mesoporous confinement range, surface chemical groups, and gradual degradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate). This achieves the slow release of a drug for as long as possible. We implanted the drug-loaded compound scaffold into New Zealand rabbits’ femur defect model to study the in vivo drug release performance and osteogenic ability. The in vivo release of isoniazid and rifampicin from the prepared composites could be effectively sustained for 12 weeks in local tissues, whereas these drugs were sustained for just 2 weeks in a control group. The blood drug concentrations were very low and most concentrations were below 5 μg/ml. Therefore, the systemic toxic adverse effect is very low. In addition, the composite exhibits good osteogenic potential in a rabbit bone defect model. The results of this study indicate that this composite has great potential for treating osteoarticular TB.     

Parathyroid Hormone Derivative with Reduced Osteoclastic Activity Promoted Bone Regeneration via Synergistic Bone Remodeling and Angiogenesis

Osteogenesis, osteoclastogenesis, and angiogenesis are the most important processes in bone repair. Parathyroid hormone (PTH) has pro‐osteogenic, pro‐osteoclastogenic, and proangiogenic effects and may be a candidate for use in bone defect repair. However, the local application of PTH to bone defects is counterproductive due to its excessive osteoclastic and bone resorptive effects. In this study, a PTH derivative, PTHrP‐2, is developed that can be applied to local bone defects. First, a modified peptide with a calcium‐binding repeat glutamine tail undergoes controlled local release from a ceramic material and is shown to be a better fit for the repair process than the unmodified peptide. Second, the modified peptide is shown to have strong pro‐osteogenic activity due to mineralization and its facilitation of serine (Ser) phosphorylation. Third, the modified peptide is shown to maintain the pro‐osteoclastogenic and proangiogenic properties of the unmodified peptide, but its pro‐osteoclastogenic activity is reduced compared to that of the unmodified peptide. The reduced pro‐osteoclastogenic and increased pro‐osteogenic properties of the modified peptide reverse the imbalance between osteoblasts and osteoclasts with local PTH application and shift bone resorption to bone regeneration.     

Triphasic ceramic coated hydroxyapatite as a niche for goat stem cell-derived osteoblasts for bone regeneration and repair

Current treatment strategies for the repair or replacement of bone use synthetic implants with stem cells and their progeny––a new approach to address unmet medical needs. This study has evaluated the effect of a silica-coated bioactive ceramic, namely HASi in comparison to hydroxyapatite (HA) on the adhesion, proliferation and osteogenic differentiation of goat bone marrow-derived mesenchymal stem cells in vitro in a prolonged culture of 28 days. The cellular activities were significantly enhanced on HASi signifying the role of silica to stimulate osteoblast cells. The fabrication of such a ‘cell-ceramic construct using autologous MSCs’ is aimed for the transplantation to a large bone defect site in the goat femur model which still remains a formidable challenge in Orthopedic surgery.     

The healing of critical-size calvarial bone defects in rat with rhPDGF-BB, BMSCs, and β-TCP scaffolds

Platelet-derived growth factor-BB (PDGF-BB) plays important roles in regenerating damaged tissue. In this study we investigated the effects of a tissue-engineered bone combined with recombinant human PDGF-BB (rhPDGF-BB), bone marrow stem cells (BMSCs) and β–tricalcium phosphate (β-TCP) to repair critical-size calvarial bone defects in rat. Proliferation and osteogenic differentiation of BMSCs treated with different concentration rhPDGF-BB (0, 10, and 50 ng/ml) was evaluated by MTT, alkaline phosphatase (ALP) activity, alizarin red staining and real-time quantitative PCR (RT-qPCR) analysis of osteogenic gene. BMSCs were then combined with rhPDGF-BB-loaded β-TCP and transplanted into 5 mm calvarial bone defects. The new bone formation and mineralization was evaluated by micro-computerized tomography (Micro-CT) and histological analysis at week 8 after operation. It was observed that the proliferation of BMSCs treated with rhPDGF-BB was enhanced with a time- and dose- dependent manner. There were increased ALP activity, mineralized deposition and elevated mRNA levels of osteogenic gene for BMSCs treated with rhPDGF-BB, particularly in the 50 ng/ml group. Histological analysis showed new bone formation and mineralization in the rhPDGF-BB/BMSCs/β-TCP group was significantly higher than BMSCs/β-TCP, rhPDGF-BB/β-TCP, and β-TCP alone group (P < 0.05). In conclusion, rhPDGF-BB/BMSCs/β-TCP is a promising tissue-engineered bone for craniofacial bone regeneration.     

Repair of rabbit femoral condyle bone defects with injectable nanohydroxyapatite/chitosan composites

Repair of massive bone loss remains a challenge to the orthopaedic surgeons. Autologous and allogenic bone grafts are choice for bone reconstructive surgery, but limited availability, risks of transmittable diseases and inconsistent clinical performances have prompted the development of tissue engineering. In the present work, the bone regeneration potential of nanohydroxyapatite/chitosan composite scaffolds were compared with pure chitosan scaffolds when implanted into segmental bone defects in rabbits. Critical size bone defects (6 mm diameter, 10 mm length) were created in the left femoral condyles of 43 adult New Zealand white rabbits. The femoral condyle bone defects were repaired by nanohydroxyapatite/chitosan compositions, pure chitosan or left empty separately. Defect-bridging was detected by plain radiograph and quantitative computer tomography at eight and 12 weeks after surgery. Tissue samples were collected for gross view and histological examination to determine the extent of new bone formation. Eight weeks after surgery, more irregular osteon formation was observed in the group treated with nanohydroxyapatite/chitosan composites compared with those treated with pure chitosan. 12 weeks after surgery, complete healing of the segmental bone defect was observed in the nanohydroxyapatite/chitosan-group, while the defect was still visible in the chitosan-group, although the depth of the defect had diminished. These observations suggest that the injectable nanohydroxyapatite/chitosan scaffolds are potential candidate materials for regeneration of bone loss.     

Interactions of total bone marrow cells with increasing quantities of macroporous calcium phosphate ceramic granules

The biological properties of synthetic calcium phosphate bioceramics have made them the third choice of material for bone reconstructive surgery, after autologous bone and allografts. Nevertheless, bioceramics lack the osteogenic properties that would allow them to repair large bone defects. One strategy in bone tissue engineering consists of associating a synthetic scaffold with osteogenic cells. Mesenchymal stem cells (MSC) are usually isolated from bone marrow cultured for several weeks and seeded on to a small quantity of bioceramic. We have studied the association of total bone marrow cells, harvested from femurs of rats, with increasing amounts of calcium phosphate ceramic granules (50–250 mg). A cell viability test indicated that a little quantity of bioceramics granules (50 mg) was less detrimental for culturing 1 million nucleated cells from the whole bone marrow population. Cell morphology, viability, adhesion and differentiation were studied after different culture periods. Among the heterogeneous population of bone marrow cells, only a limited amount of cells attached and differentiated on the bioceramics. To explain the influence of the amount of synthetic scaffold on cell viability, media calcium concentrations were measured. Low cell viability could be explained by calcium phosphate precipitation leading to a decrease in calcium concentrations observed with relatively large amounts of scaffold. This study showed that the chemical stability of the ceramic plays a critical role in the viability of bone marrow cells.     

A silk fibroin/chitosan scaffold in combination with bone marrow-derived mesenchymal stem cells to repair cartilage defects in the rabbit knee

Bone marrow-derived mesenchymal stem cells (BMSCs) were seeded in a three-dimensional scaffold of silk fibroin (SF) and chitosan (CS) to repair cartilage defects in the rabbit knee. Totally 54 rabbits were randomly assigned to BMSCs + SF/CS scaffold, SF/CS scaffold and control groups. A cylindrical defect was created at the patellofemoral facet of the right knee of each rabbit and repaired by scaffold respectively. Samples were prepared at 4, 8 and 12 weeks post-surgery for gross observation, hematoxylin–eosin and toluidine blue staining, type II collagen immunohistochemistry, Wakitani histology. The results showed that differentiated BMSCs proliferated well in the scaffold. In the BMSCs + SF/CS scaffold group, the bone defect was nearly repaired, the scaffold was absorbed and immunohistochemistry was positive. In the SF/CS scaffold alone group, fiber-like tissues were observed, the scaffold was nearly degraded and immunohistochemistry was weakly positive. In the control group, the defect was not well repaired and positive immunoreactions were not detected. Modified Wakitani scores were superior in the BMSCs + SF/CS scaffold group compared with those in other groups at 4, 8 and 12 weeks (P < 0.05). A SF/CS scaffold can serve as carrier for stem cells to repair cartilage defects and may be used for cartilage tissue engineering.     

In vitro evaluation of bioactive strontium-based ceramic with rabbit adipose-derived stem cells for bone tissue regeneration

The development of bone replacement materials is an important objective in the field of orthopaedic surgery. Due to the drawbacks of treating bone defects with autografts, synthetic bone graft materials have become optional. So in this work, a bone tissue engineering approach with radiopaque bioactive strontium incorporated calcium phosphate was proposed for the preliminary cytocompatibility studies for bone substitutes. Accumulating evidence indicates that strontium containing biomaterials promote enhanced bone repair and radiopacity for easy imaging. Hence, strontium calcium phosphate (SrCaPO₄) and hydroxyapatite scaffolds have been investigated for its ability to support and sustain the growth of rabbit adipose-derived mesenchymal stem cells (RADMSCs) in vitro. They were characterized via Micro-CT for pore size distribution. Cells used were isolated from New Zealand White rabbit adipose tissue, characterized by FACS and via differentiation into the osteogenic lineage by alkaline phosphatase, Masson’s trichome, Alizarin Red and von Kossa staining on day 28. Material-cell interaction was observed by SEM imaging of cell morphology on contact with material. Live–Dead analysis was done by confocal laser scanning microscopy and cell cluster analysis via μCT. The in vitro biodegradation, elution and nucleation of apatite formation of the material was evaluated using simulated body fluid and phosphate buffered saline in static regime up to 28 days at 37 °C. These results demonstrated that SrCaPO₄ is a good candidate for bone tissue engineering applications and with osteogenically-induced RADMSCs, they may serve as potential implants for the repair of critical-sized bone defects.     

Ability of polyurethane foams to support placenta-derived cell adhesion and osteogenic differentiation: preliminary results

In bone tissue reconstruction, the use of engineered constructs created by mesenchymal stem cells (MSCs) that differentiate and proliferate into 3D porous scaffolds is an appealing alternative to clinical therapies. Human placenta represents a possible source of MSCs, as it is readily available without invasive procedures and because of the phenotypic plasticity of many of the cell types isolated from this tissue. The scaffold considered in this work is a slowly degradable polyurethane foam (EF PU foam), synthesized and characterized for morphology and in vitro interaction with chorion mesenchymal cells (CMCs). These cells were isolated from human term placenta and cultured onto the EF PU foam using two different culture media (EMEM and NH osteogenic differentiation medium). Synthesized EF PU foam showed homogeneous pore size and distribution, with 89% open porosity. In vitro tests showed CMCs scaffold colonization, as confirmed by Scanning Electron Microscopy (SEM) observations and hematoxylin–eosin staining. Alizarin Red staining revealed the presence of a small amount of calcium deposition for the samples treated with the osteogenic differentiation medium. Therefore, the proposed EF PU foam appears to stimulate cell adhesion in vitro, sustaining CMCs growth and differentiation into the osteogenic lineage.     

Synthesis, characterization and osteoconductivity properties of bone fillers based on alendronate-loaded poly(ε-caprolactone)/hydroxyapatite microspheres

A superior drug controlled release system capable of achieving efficient osteogenesis is in imperative demand because of limited bone substitute tissue for the treatment of bone defect. In the present study, we investigated the potential of using poly(ε-caprolactone)–hydroxyapatite (PCL–HA) composite microspheres as an injectable bone repair vehicle by controlled release of alendronate (AL), a medicine that belongs to the bisphosphonates family. The PCL/HA–AL microspheres were prepared with solid/oil/water emulsion technique, which included two processes: (1) AL was loaded on the hydroxyapatite nanoparticles; (2) the HA–AL complex was built in the PCL matrix. The spherical PCL/HA–AL microspheres were characterized with its significantly improved encapsulation efficiency of hydrophilic AL and better sustained release. Human bone mesenchymal stem cells (hMSCs) were cultured on the surface of these microspheres and exhibited high proliferative profile. Specifically, in osteogenic medium, hMSCs on the surface of PCL/HA–AL microspheres displayed superior osteogenic differentiation which was verified by alkaline phosphatase activity assay. In conclusion, by presenting strong osteogenic commitment of hMSCs in vitro, the PCL/HA–AL microspheres have the potential to be used as an injectable vehicle for local therapy of bone defect.     

Poloxamer-based hydrogels hardening at body core temperature as carriers for cell based therapies: in vitro and in vivo analysis

Cell-based regenerative therapies for bone defects usually employ bone precursor cells seeded on solid scaffolds. Thermosensitive hydrogels that harden at body core temperature are promising alternative cell carriers as they are applicable minimally invasively. We modified Pluronic^® P123 with different chain extenders and assessed rheology and biocompatibility of the resulting hydrogels. The best candidate was tested in a rat’s femoral defect model. All gels hardened above 25 °C with butane-diisocyanate-hydrogels (BDI-gels) displaying the highest storage moduli. BDI-gels showed the most favourable biocompatibility and did not affect cellular adipogenic or osteogenic differentiation in vitro. Implantation of BDI-hydrogel into femoral defects did not impede bone healing in vivo as evidenced by μCT and histological analysis. We conclude that thermosensitive BDI-gels are promising alternative cell carriers. The gels harden upon injection in vivo without interfering with bone metabolism. Further experiments will assess the gels’ capacity to effectively transport living cells into bone defects.     

Osteoinduction of calcium phosphate biomaterials in small animals

Although osteoinduction mechanism of calcium phosphate (CP) ceramics is still unclear, several essential properties have been reported, such as chemical composition, pore size and porosity, etc. In this study, calcium phosphate powder (Ca₃(PO₄)₂, CaP, group 1), biphasic calcium phosphate ceramic powder (BCP, group 2), and intact BCP rods (group 3) were implanted into leg muscles of mice and dorsal muscles of rabbits. One month and three months after implantation, samples were harvested for biological and histological analysis. New bone tissues were observed in 10/10 samples in group 1, 3/10 samples in group 2, and 9/10 samples in group 3 at 3rd month in mice, but not in rabbits. In vitro, human mesenchymal stem cells (hMSCs) were cultured with trace CaP and BCP powder, and osteogenic differentiation was observed at day 7. Our results suggested that chemical composition is the prerequisite in osteoinduction, and pore structure would contribute to more bone formation.     

A hydrophylic polymer system enhanced articular cartilage regeneration in vivo

This study describes a new method for the repair of large articular cartilage defects in the knee joint and compares the effect of two polymer systems on the quality of the repair tissue. The two systems are a newly developed hydrophylic system, based on poly-ethyl-methacrylate (PEMA) polymer and tetra-hydro-furfuryl-methacrylate (THFMA) monomer and the conventional bone cement polymer system, based on poly-methyl-methacrylate (PMMA) polymer and methyl-methacrylate (MMA) monomer. Thirty adult Sandy-lop rabbits were used. Both knees were operated on in each animal, the one defect received either PEMA/THFMA or conventional bone cement and the contralateral defect received no biomaterial (control group). Femora were retrieved at six weeks and the repair tissue was studied by histology, histochemistry and immuno-histochemistry. PEMA/THFMA enhanced the quality of the repair significantly (p<0.0001). By six weeks hyaline-like articular cartilage was the predominant tissue covering the defects and it was fully integrated with the surrounding normal articular cartilage. Immuno-localization showed cartilage components, including collagen type II, distributed evenly throughout its matrix. PMMA/MMA on the other hand did not improve significantly the repair tissue, which was predominately fibro-cartilaginous, poorly bonded to the adjacent normal articular cartilage. The method of implantation is simple and easily reproducible and the new polymer has been well-accepted by the rabbits.