Ectopic Osteoid and Bone Formation by Three Calcium-Phosphate Ceramics in Rats,Rabbits and Dogs

Calcium phosphate ceramics with specific physicochemical properties have been shown to induce de novo bone formation upon ectopic implantation in a number of animal models. In this study we explored the influence of physicochemical properties as well as the animal species on material-induced ectopic bone formation. Three bioceramics were used for the study: phase-pure hydroxyapatite (HA) sintered at 1200°C and two biphasic calcium phosphate (BCP) ceramics, consisting of 60 wt.% HA and 40 wt.% TCP (β-Tricalcium phosphate), sintered at either 1100°C or 1200°C. 108 samples of each ceramic were intramuscularly implanted in dogs, rabbits, and rats for 6, 12, and 24 weeks respectively. Histological and histomorphometrical analyses illustrated that ectopic bone and/or osteoid tissue formation was most pronounced in BCP sintered at 1100°C and most limited in HA, independent of the animal model. Concerning the effect of animal species, ectopic bone formation reproducibly occurred in dogs, while in rabbits and rats, new tissue formation was mainly limited to osteoid. The results of this study confirmed that the incidence and the extent of material-induced bone formation are related to both the physicochemical properties of calcium phosphate ceramics and the animal model.     

Hydroxyapatite from Cuttlefish Bone: Isolation,Characterizations, and Applications

Hydroxyapatite (HA), a bioceramic, is a widely utilized material for bone tissue repair and regeneration because of its excellent properties such as biocompatibility, exceptional mechanical strength, and osteoconductivity. HA can be obtained by both synthetic and natural means. Animal bones are often considered a promising natural resource for the preparation of pure HA for biological and biomedical applications. Cuttlefish bone, also called as cuttlebone, mainly consists of calcium carbonate, and pure HA can be produced by adding phosphoric acid or ammonium hydrogen phosphate to it. Recently, cuttlefish bone-derived HA has shown promising results in terms of bone tissue repair and regeneration. The synthesized cuttlefish bone-derived has shown excellent biocompatibility, cell proliferation, increased alkaline phosphate activity, and efficient biomineralization ability with mesenchymal stem cells and osteoblastic cells. To further improve the biological properties of cuttlefish bone-derived HA, bioglass, polycaprolactone, and polyvinyl alcohol were added to it, which gave better results in terms of cell proliferation and osteogenic differentiation. Cuttlefish bone-derived HA with polymeric substances provides excellent bone formation under in vivo conditions. The studies indicate that cuttlefish bone-derived HA, along with polymeric and, protein materials, will be promising biomaterials in the field of bone tissue regeneration.     

Repair of rabbit ulna segmental bone defect using freshly isolated adipose-derived stromal vascular fraction

Background aimsStromal vascular fractions (SVF) from adipose tissue have heterogeneous cell populations, and include multipotent adipose-derived stem cells. The advantages of using of SVF include the avoidance of an additional culture period, a reduced risk of extensive cell contamination, and cost-effectiveness.MethodsUnilateral 20-mm mid-diaphyseal segmental defects in rabbit ulna were treated with one of the following: polylactic glycolic acid (PLGA) scaffold alone (group 1, control), a PLGA scaffold with undifferentiated SVF cells (group 2), or a PLGA scaffold with osteogenically differentiated SVF cells (group 3). At 8 weeks after implantation, five rabbits in each treatment group were killed to assess bone defect healing by plain radiography, quantitative microcomputed tomography and histology.ResultsThe SVF cells were well grown on PLGA scaffolds and expressed type I collagen and alkaline phosphatase (ALP). The intensity of ALP and OPN gene expressions in osteogenic medium culture were increased from 14 days to 28 days. In vivo evaluations at 8 weeks showed that treatment of SVF cells with or without osteogenic differentiation resulted in more bone formation in the critically sized segmental defects than PLGA scaffold alone. Osteogenically differentiated SVF cells significantly enhanced bone healing compared with undifferentiated SVF cells.ConclusionsAdipose-derived stromal SVF showed osteogenic potential in vitro. Accordingly, SVF could provide a cell source for bone tissue engineering. However, treatment with uncultured SVF cells on bone healing was not satisfactory in the in vivo animal model.     

Dexamethasone-loaded hydroxyapatite enhances bone regeneration in rat calvarial defects

A combination of bioceramics and osteogenic factors is potentially useful for bone regeneration applications. In the present study, hydroxyapatite particles (HA) were loaded with dexamethasone (Dex) and then characterized using SEM and drug release study. The bone regeneration ability of Dex-loaded HA (Dex/HA) was investigated in a rat critical size bone defect using digital mammography, multislice spiral-computed tomography (MSCT) imaging, and histological analysis. The HA and Dex/HA showed nano and micro-scale morphology with a nearly homogenous distribution of diameter. In addition, about 90 % of the drug was released from Dex/HA over a period of three days. After 8 weeks of implantation in rat calvarial defects, no sign of inflammation or complication was observed at the site of surgery. According to digital mammography and MSCT, Dex/HA showed the highest bone regeneration in rat bone defects compared to those received drug-free HA. Histological studies confirmed these data and showed osteointegration to the surrounding tissue. Taking all together, it was demonstrated that Dex/HA can be used as an appropriate synthetic graft for bone tissue engineering applications. These newly developed bioceramics can be used as new bone graft substitutes in orthopaedic surgery and is capable of enhancing bone regeneration.     

Combination of Bioactive Polymeric Membranes and Stem Cells for Periodontal Regeneration: In Vitro and In Vivo Analyses

Regeneration of periodontal tissues requires a concerted effort to obtain consistent and predictable results in vivo. The aim of the present study was to test a new family of bioactive polymeric membranes in combination with stem cell therapy for periodontal regeneration. In particular, the novel polyester poly(isosorbide succinate-co-L-lactide) (PisPLLA) was compared with poly(L-lactide) (PLLA). Both polymers were combined with collagen (COL), hydroxyapatite (HA) and the growth factor bone morphogenetic protein-7 (BMP7), and their osteoinductive capacity was evaluated via in vitro and in vivo experiments. Membranes composed of PLLA/COL/HA or PisPLLA/COL/HA were able to promote periodontal regeneration and new bone formation in fenestration defects in rat jaws. According to quantitative real-time polymerase chain reaction (qRT-PCR) and Alizarin Red assays, better osteoconductive capacity and increased extracellular mineralization were observed for PLLA/COL/HA, whereas better osteoinductive properties were associated with PisPLLA/COL/HA. We concluded that membranes composed of either PisPLLA/COL/HA or PLLA/COL/HA present promising results in vitro as well as in vivo and that these materials could be potentially applied in periodontal regeneration.     

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.     

Ectopic Osteogenesis of Macroscopic Tissue Constructs Assembled from Human Mesenchymal Stem Cell-Laden Microcarriers through In Vitro Perfusion Culture

We had previously demonstrated the feasibility of preparing a centimeter-sized bone tissue construct by following a modular approach. In the present study, the objectives were to evaluate osteogenesis and tissue formation of human amniotic mesenchymal stem cells-laden CultiSpher S microcarriers during in vitro perfusion culture and after subcutaneous implantation. Microtissues were prepared in dynamic culture using spinner flasks in 28 days. In comparison with 1-week perfusion culture, microtissues became more obviously fused, demonstrating significantly higher cellularity, metabolic activity, ALP activity and calcium content while maintaining cell viability after 2-week perfusion. After subcutaneous implantation in nude mice for 6 and 12 weeks, all explants showed tight contexture, suggesting profound tissue remodeling in vivo. In addition, 12-week implantation resulted in slightly better tissue properties. However, in vitro perfusion culture time exerted great influence on the properties of corresponding explants. Degradation of microcarriers was more pronounced in the explants of 2-week perfused macrotissues compared to those of 1-week perfusion and directly implanted microtissues. Moreover, more blood vessel infiltration and bone matrix deposition with homogeneous spatial distribution were found in the explants of 2-week perfused macrotissues. Taken together, in vitro perfusion culture time is critical in engineering bone tissue replacements using such a modular approach, which holds great promise for bone regeneration.     

Comparative in vitro study regarding the biocompatibility of titanium-base composites infiltrated with hydroxyapatite or silicatitanate

Background

The development of novel biomaterials able to control cell activities and direct their fate is warranted for engineering functional bone tissues. Adding bioactive materials can improve new bone formation and better osseointegration. Three types of titanium (Ti) implants were tested for in vitro biocompatibility in this comparative study: Ti6Al7Nb implants with 25% total porosity used as controls, implants infiltrated using a sol–gel method with hydroxyapatite (Ti HA) and silicatitanate (Ti SiO₂). The behavior of human osteoblasts was observed in terms of adhesion, cell growth and differentiation.

Results

The two coating methods have provided different morphological and chemical properties (SEM and EDX analysis). Cell attachment in the first hour was slower on the Ti HA scaffolds when compared to Ti SiO₂ and porous uncoated Ti implants. The Alamar blue test and the assessment of total protein content uncovered a peak of metabolic activity at day 8–9 with an advantage for Ti SiO₂ implants. Osteoblast differentiation and de novo mineralization, evaluated by osteopontin (OP) expression (ELISA and immnocytochemistry), alkaline phosphatase (ALP) activity, calcium deposition (alizarin red), collagen synthesis (SIRCOL test and immnocytochemical staining) and osteocalcin (OC) expression, highlighted the higher osteoconductive ability of Ti HA implants. Higher soluble collagen levels were found for cells cultured in simple osteogenic differentiation medium on control Ti and Ti SiO₂ implants. Osteocalcin (OC), a marker of terminal osteoblastic differentiation, was most strongly expressed in osteoblasts cultivated on Ti SiO₂ implants.

Conclusions

The behavior of osteoblasts depends on the type of implant and culture conditions. Ti SiO₂ scaffolds sustain osteoblast adhesion and promote differentiation with increased collagen and non-collagenic proteins (OP and OC) production. Ti HA implants have a lower ability to induce cell adhesion and proliferation but an increased capacity to induce early mineralization. Addition of growth factors BMP-2 and TGFβ1 in differentiation medium did not improve the mineralization process. Both types of infiltrates have their advantages and limitations, which can be exploited depending on local conditions of bone lesions that have to be repaired. These limitations can also be offset through methods of functionalization with biomolecules involved in osteogenesis.

     

Self-healing and injectable hybrid hydrogel for bone regeneration of femoral head necrosis and defect

Background

HA modified by bisphosphonate (BP) (HA-BP) was synthesized by chemical reaction and possessed promising properties such as self-healing, injection ability, and strong adhesion. The main aim of this study was to confirm its role in promoting osteogenic differentiation in vitro and bone regeneration in vivo.

The Improved Biological Performance of a Novel Low Elastic Modulus Implant

Background

The mismatch of the elastic modulus between implants and bone tissue can lead to stress shielding, bone resorption and poor osseointegration. Compared with normal bone tissue, this problem is much more serious in osteoporosis. The purpose of this study was designed to find out whether the novel Ti-24Nb-4Zr-7.9Sn (TNZS) implant with low elastic modulus and high strength was suitable for biomedical material, especially in osteoporosis.

Methodology

In vitro study, the viability and Alkaline phosphatase (ALP) activity of osteoblasts on the TNZS and Ti-6V-4V (TAV) were observed. In vivo study, 30 adult female New Zealand rabbits were selected and divided randomly into two groups: sham-operation (SHAM, n = 6) and ovariectomised in combination with methylprednisolone treatment (OVX+MP, n = 24). Two implants were then placed in the tibia of each OVX + MP group rabbit, one in each side (left: TAV; right: TNZS). The OVX + MP group rabbits were sacrificed at 4 and 12 weeks after the implantation. The osteoporotic bone responses to the TNZS and TAV implants were evaluated by pull-out test, Micro-CT analyses and histological observation.

Principal Findings

Compared with the TAV group, the TNZS group showed a significant increase (P<0.05) in cell viability and ALP activity, new bone formation and pull-out force.

Conclusions

The novel TNZS implants show good biological performance both in vitro and in vivo, which suggests that the alloys are suitable for biomedical applications, especially in osteoporosis.     

Simple Signaling Molecules for Inductive Bone Regenerative Engineering

With greater than 500,000 orthopaedic procedures performed in the United States each year requiring a bone graft, the development of novel graft materials is necessary. We report that some porous polymer/ceramic composite scaffolds possess intrinsic osteoinductivity as shown through their capacity to induce in vivo host osteoid mineralization and in vitro stem cell osteogenesis making them attractive synthetic bone graft substitutes. It was discovered that certain low crystallinity ceramics partially dissociate into simple signaling molecules (i.e., calcium and phosphate ions) that induce stem cells to endogenously produce their own osteoinductive proteins. Review of the literature has uncovered a variety of simple signaling molecules (i.e., gases, ions, and redox reagents) capable of inducing other desirable stem cell differentiation through endogenous growth factor production. Inductive simple signaling molecules, which we have termed inducerons, represent a paradigm shift in the field of regenerative engineering where they can be utilized in place of recombinant protein growth factors.     

In vitro prominent bone regeneration by release zinc ion from Zn-modified implant

Zinc is one of the trace elements which induce the proliferation and the differentiation of the osteoblast. In the previous study, we found that zinc ions (Zn²⁺ ion)-releasing titanium implants had excellent bone fixation using a rabbit femurs model. In this study, we isolated the Zn²⁺ ions (eluted Zn²⁺ ion; EZ) released from the implant surface, and evaluated the effect of EZ on the osteogenesis of human bone marrow-derived mesenchymal cells (hBMCs). In the result, it was found that the EZ stimulated cell viability, osteoblast marker gene (type I collagen, osteocalcin (OC), alkaline phosphatase (ALP) and bone sialoprotein (BSP)) expressions and calcium deposition in hBMCs.     

Ion beam treatment of titanium surfaces for enhancing deposition of hydroxyapatite from solution

Surface coating with hydroxyapatite (HA) is a common way to improve the osseointegration of orthopaedic and dental titanium (Ti)-based materials. The main problems with current techniques are changes in composition during heating and poor adhesion to the surface. An alternative method is deposition of HA onto an activated surface out of a solution. The present work studies the surface treatment involving ion implantation of Na into Ti to induce a modification in chemistry and morphology, showing sodium titanate (Na(2)TiO(3)) incorporated within the surface layer with concentration, depth distribution, and morphology depending on the parameters of the ion implantation. Such ion-implanted Ti surfaces actively induce heterogeneous precipitation of HA from a simulated body fluid containing physiological concentrations of calcium and phosphate ions. This is compared with the activation by NaOH etching. The growth of bone forming cells on the pure Na implanted surface is oriented without an increased bone formation. Cell growth on the NaOH etched surface is reduced. After deposition of HA on both surfaces cell the growth pattern was improved.     

In vitro and in vivo induction of bone formation based on ex vivo gene therapy using rat adipose-derived adult stem cells expressing BMP-7

BACKGROUND: Adipose-derived adult stem (ADAS) cells are multipotent cells capable of differentiating into osteoblasts, adipocytes and chondrocytes. The aim of this study was to determine whether BMP-7-expressing ADAS cells would elicit bone formation invitro and in vivo. METHODS: ADAS cells were harvested from Lewis rats and transduced with adenovirus carrying the recombinant human bone morphogenetic protein-7 (Ad-BMP-7) gene. Untransduced cells and cells transduced with adenovirus carrying the enhanced green fluorescence protein (Ad-EGFP) gene served as controls. BMP-7 expression was assessed by RT-PCR, immunofluorescence on day 1, and Western blot on days 4, 8 and 12. Alkaline phosphatase (ALP) activity was assayed on days 2, 4, 6, 8, 10 and 12. Osteocalcin production and bone nodule formation were detected by immunohistochemistry and von Kossa stain on day 12. A total of 1 x 10(6) cells mixed with type I collagen were implanted into the subcutaneous pocket in Lewis rat and subjected to histologic analysis 1, 2 and 4 weeks post-implantation. RESULTS: The Ad-BMP-7-transduced ADAS cells expressed BMP-7 at both mRNA and protein levels. ALP activity was detected in Ad-BMP-7-transduced cells from day 2 to day 12, peaking on day 8. Osteocalcin production and matrix mineralization further confirmed that these cells differentiated into osteoblasts and induced bone formation in vitro. Histologic examination revealed that implantation of BMP-7-expressing ADAS cells could induce new bone formation in vivo. DISCUSSION: ADAS cells would be a promising source of adult autologous stem cells for BMP gene therapy and tissue engineering.     

In vitro and in vivo tests of hydrothermally synthesised hydroxyapatite coating

High pure and crystalline Hydroxyapatite (HA) coatings on titanium alloy were prepared by hydrothermal synthesis (HS) of plasma-sprayed (PS) precursors from brushite powders (HS-HA). In vitro and in vivo tests were done to evaluate its biological property. The HS-HA coating was compared with the current PS-HA coating. Cultures of the primary osteoblasts on these two HA coatings showed similar cell attachment, proliferation and alkaline phosphatase (ALP) expression. The cell morphology on the coatings was demonstrated by scanning electron microscopy (SEM). The cell spread well at 1 day after seeding culture and the extracellular matrix was secreted after 14 days culture. Histomorphometric analysis was conducted on samples implanted in femoral bone of four dogs for 1 and 3 months, and bone-implant contact percentage was evaluated by light microscopy. The calcium and phosphate distribution on the interface of bone-implant was analysed by SEM and electron dispersive X-ray (EDX) analysis. The results show the osteoconduction of HS-HA coated implants.     

Bortezomib enhances the osteogenic differentiation capacity of human mesenchymal stromal cells derived from bone marrow and placental tissues

Bortezomib (BZB) is a chemotherapeutic agent approved for treating multiple myeloma (MM) patients. In addition, there are several reports showing that bortezomib can induce murine mesenchymal stem cells (MSCs) to undergo osteogenic differentiation and increase bone formation in vivo. MSCs are the multipotent stem cells that have capacity to differentiate into several mesodermal derivatives including osteoblasts. Nowadays, MSCs mostly bone marrow derived have been considered as a valuable source of cell for tissue replacement therapy. In this study, the effect of bortezomib on the osteogenic differentiation of human MSCs derived from both bone marrow (BM-MSCs) and postnatal sources such as placenta (PL-MSCs) were investigated. The degree of osteogenic differentiation of BM-MSCs and PL-MSCs after bortezomib treatment was assessed by alkaline phosphatase (ALP) activity, matrix mineralization by Alizarin Red S staining and the expression profiles of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP. The results showed that 1 nM and 2 nM BZB can induce osteogenic differentiation of BM-MSCs and PL-MSCs as demonstrated by increased ALP activity, increased matrix mineralization and up-regulation of osteogenic differentiation marker genes, Osterix, RUNX2 and BSP as compared to controls. The enhancement of osteogenic differentiation of MSCs by bortezomib may lead to the potential therapeutic applications in human diseases especially patients with osteopenia.     

Integration of Stem Cell to Chondrocyte-Derived Cartilage Matrix in Healthy and Osteoarthritic States in the Presence of Hydroxyapatite Nanoparticles

We investigated the effectiveness of integrating tissue engineered cartilage derived from human bone marrow derived stem cells (HBMSCs) to healthy as well as osteoarthritic cartilage mimics using hydroxyapatite (HA) nanoparticles immersed within a hydrogel substrate. Healthy and diseased engineered cartilage from human chondrocytes (cultured in agar gels) were integrated with human bone marrow stem cell (HBMSC)-derived cartilaginous engineered matrix with and without HA, and evaluated after 28 days of growth. HBMSCs were seeded within photopolymerizable poly (ethylene glycol) diacrylate (PEGDA) hydrogels. In addition, we also conducted a preliminary in vivo evaluation of cartilage repair in rabbit knee chondral defects treated with subchondral bone microfracture and cell-free PEGDA with and without HA. Under in vitro conditions, the interfacial shear strength between tissue engineered cartilage derived from HBMSCs and osteoarthritic chondrocytes was significantly higher (p < 0.05) when HA nanoparticles were incorporated within the HBMSC culture system. Histological evidence confirmed a distinct spatial transition zone, rich in calcium phosphate deposits. Assessment of explanted rabbit knees by histology demonstrated that cellularity within the repair tissues that had filled the defects were of significantly higher number (p < 0.05) when HA was used. HA nanoparticles play an important role in treating chondral defects when osteoarthritis is a co-morbidity. We speculate that the calcified layer formation at the interface in the osteoarthritic environment in the presence of HA is likely to have attributed to higher interfacial strength found in vitro. From an in vivo standpoint, the presence of HA promoted cellularity in the tissues that subsequently filled the chondral defects. This higher presence of cells can be considered important in the context of accelerating long-term cartilage remodeling. We conclude that HA nanoparticles play an important role in engineered to native cartilage integration and cellular processes.     

冲击波诱导人骨髓基质细胞体内成骨研究

为了研究冲击波(SW)诱导人骨髓基质细胞(hMSCs)在动物体内成骨作用,根据前期工作结果,应用适宜能量冲击波(10kV,500次)处理体外培养的hMSCs,将SW组和对照组hMSCs与羟基磷灰石(HA)载体复合后体外培养2周,应用扫描电镜(SEM)检测细胞在载体表面的生长情况.将hMSCs-HA载体复合体植入裸鼠皮下,分别于术后4周、8周取材进行组织学、四环素荧光标记、SEM观察、碱性磷酸酶测定、RT-PCR检测骨钙素mRNA表达.结果表明,SW组及对照组细胞与HA载体体外复合后生长良好,且SW组细胞分泌较多的细胞基质;细胞载体复合体植入动物体内后,SW组载体表面有类骨组织形成,而对照组HA载体表面无骨组织形成;SW组与对照组的hMSCs-HA载体复合体碱性磷酸酶表达有显著性差异(P<0.01);SW组hMSCs-HA载体复合体术后4周与8周表达骨钙素mRNA,而对照组则无表达.提示hMSCs经适宜能量冲击波作用后与HA载体复合植入裸鼠体内具有成骨作用,适宜能量的冲击波作为一种新的促进hMSCs成骨分化的方法,可应用于组织工程领域.     

Bioceramic-collagen scaffolds loaded with human adipose-tissue derived stem cells for bone tissue engineering

The combination of bioceramics and stem cells has attracted the interest of research community for bone tissue engineering applications. In the present study, a combination of Bio-Oss^® and type 1 collagen gel as scaffold were loaded with human adipose-tissue derived mesenchymal stem cells (AT-MSCs) after isolation and characterization, and the capacity of them for bone regeneration was investigated in rat critical size defects using digital mammography, multi-slice spiral computed tomography imaging and histological analysis. 8 weeks after implantation, no mortality or sign of inflammation was observed in the site of defect. According to the results of imaging analysis, a higher level of bone regeneration was observed in the rats receiving Bio-Oss^®-Gel compared to untreated group. In addition, MSC-seeded Bio-Oss-Gel induced the highest bone reconstruction among all groups. Histological staining confirmed these findings and impressive osseointegration was observed in MSC-seeded Bio-Oss-Gel compared with Bio-Oss-Gel. On the whole, it was demonstrated that combination of AT-MSCs, Bio-Oss and Gel synergistically enhanced bone regeneration and reconstruction and also could serve as an appropriate structure to bone regenerative medicine and tissue engineering application.