There is increasing application of bone morphogenetic proteins
(BMPs) owing to their role in promoting fracture healing and bone
fusion. However, an optimal delivery system has yet to be identified.
The aims of this study were to synthesise bioactive BMP-2, combine
it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide)
(α-TCP/PLGA) nanocomposite and study its release from the composite. BMP-2 was synthesised using an Objectives
Methods
The annual incidence of fractures in the UK is almost 4%. Bone grafting procedures and segmental bone transport have been employed for bone tissue regeneration. However, their limited availability, donor site morbidity and increased cost mean that there is still a large requirement for alternative methods and there is considerable research into regeneration using bone morphogenetic proteins (BMPs). The aims of this study are to synthesise and combine BMP-2 with a novel nanocomposite and study its release. BMP-2 was synthesised using an E. coli expression system and purified. C2C12 cells were used to test its bioactivity using an alkaline phosphatase (ALP) assay. The modified solution evaporation method was used to fabricate 30% a-TCP/PLGA nanocomposite and it was characterized using SEM, TEM, TGA, XRD, EDX and particle size analysis. The release pattern of adsorbed BMP-2 was studied using an ELISA assay.Introduction
Materials and Methods
An experimental rabbit model was used to test the null hypothesis,
that there is no difference in new bone formation around uncoated
titanium discs compared with coated titanium discs when implanted
into the muscles of rabbits. A total of three titanium discs with different surface and coating
(1, porous coating; 2, porous coating + Bonemaster (Biomet); and
3, porous coating + plasma-sprayed hydroxyapatite) were implanted
in 12 female rabbits. Six animals were killed after six weeks and
the remaining six were killed after 12 weeks. The implants with
surrounding tissues were embedded in methyl methacrylate and grinded
sections were stained with Masson-Goldners trichrome and examined
by light microscopy of coded sections.Objectives
Methods
There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides
Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in
Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and flow cytometry. The biological effects of the supernatant on cell proliferation, osteogenesis, and angiogenesis in vitro were evaluated using human mesenchymal stem cells (hMSCs) and human umbilical cord vein endothelial cells (HUVECs). Results. The supernatant contained several GFs/CKs, with especially high levels of basic fibroblast growth factor, and CD34+ cells as the stem/progenitor cell fraction. With regard to biological potential, we confirmed that cell proliferation,
Demineralized bone matrix (DBM) is a natural, collagen-based, well-established osteoinductive biomaterial. Nevertheless, there are conflicting reports on the efficacy of this product. The purpose of this study was to evaluate whether DBM collagen structure is affected by particle size and can influence DBM osteoinductivity. Sheep cortical bone was ground and particles were divided in three fractions with different sizes, defined as large (L, 1–2 mm), medium (M, 0.5–1 mm), and small (S, < 0.5 mm). After demineralization, the three DBM samples were characterized by DTA analysis, XRD, ICP-OES, and FTIR. Data clearly showed a particle size-dependent alteration in collagen structure, with DBM-M being altered but not as much as DBM-S. The in vivo study showed that only DBM-M was able to induce new bone formation in a subcutaneous ectopic mouse model. When sheep MSC were seeded onto DBM particles before implantation, all DBM particles were able to induce new bone formation with the best incidence for DBM-M and DBM-S. Gene expression analysis performed on recovered implants supports the histological results and underlines the supportive role of MSC in DBM
External fixation of distal tibial fractures is often associated with delayed union. We have investigated whether union can be enhanced by using recombinant bone morphogenetic protein-7 (rhBMP-7).
Mesenchymal Stromal Cells (MSC) are promising therapies for fracture healing. However, undifferentiated MSC may act only through an inductive paracrine effect without direct bone formation. Here, we developed an injectable product constituted of human bone-forming cells derived from bone marrow (BM)-MSC (ALLO-P2) that display more potent bone repair properties not only by stimulating host
The discussion will focus on new approaches to reduce bacterial adhesion on the surface of polymethylmethacrylate (PMMA) in contact with bone, comparing the clinical and engineering point of view. One possibility is to encourage and speed up direct interaction with the bone, for example by adding a bioactive phase in the cement (hydroxyapatite, glass and bioactive glass ceramic). A widespread strategy is also the addition of different types of antibiotics (gentamicin, tobramycin vancomycin, etc.), although they are known to have some drawbacks: not complete release, resistant strain development. Another strategy could be represented by the PMMA-based composite cements loaded with a completely inorganic filler consisting of a bioactive glass doped with ions whose bioactivity mechanism is well-known and encompasses a chemical and biological interaction with tissues promoting
Sustained release of BMP-2 is reported to be able to reduce the required dose of BMP-2 for bone induction. Nanohydroxyapatite (nHAp) has an
The current gold standard bone substitute is still autologous bone, despite the fact that its harvest demands for a second operation site, causes additional pain, discomfort, potential destruction of the grafting site, and is limited in supply. Since newly developed clinical approaches like transplantation of cells are invasive and costly, and
Long-term survival of massive prostheses used to treat bone cancers is associated with extra-cortical bone growth and osteointegration into a grooved hydroxyapatite coated collar positioned adjacent to the transection site on the implant shaft [1]. The survivorship at 10 years reduces from 98% to 75% where osteointegration of the shaft does not occur. Although current finite element (FE) methods successfully model bone adaption, optimisation of adventitious new bone growth and osteointegration is difficult to predict. There is thus a need to improve existing FE models by including biological processes of osteoconduction and
We have developed a new drug-delivery system using reconstituted bone xenograft to treat chronic osteomyelitis. This material, which has the capabilities of
Background, Context and Motivation. “Increases in reconstructive orthopaedic surgery, resulting from advances in surgical practice and the ageing population, have lead to a demand for bone graft that far exceeds supply.”…Traditional bone grafting methods have been linked with a number of negative issues including increased morbidity due to secondary operation site and action as a vector for spread of disease. (Hing 2004). A solution to these insufficiencies would be the creation of a synthetic osteoinductive bone graft material. This would vastly improve bone graft surgery success rates and expedite post-op recovery times. The aim of this study was to classify then explore the dissolution rates of three experimental hydroxyapatite/silicate apatite synthetic bonegrafts in physiological solutions, (phosphate buffered saline, (PBS) +/− serum proteins, (PBS +FCS). The overall objective being to identify whether there is an explainable significant difference in ion exchange that could be behind the osteoinductive phenomena. Methods Used. Classification of the apatite samples, (HA, SA1 and SA2), was conducted via X-Ray diffraction, FTIR-PAS Spectroscopy and SEM with EDS analysis. A dissolution experiment of the experimental apatites was conducted in PBS and PBS + FCS solutions, over time periods of 1, 2 and 4 hours, and at 1, 2, 4 and 8 days, with repeat measures. Results and Conclusions. Silicon both free in solution and at the apatite surface was found to be key for
Summary Statement. The problem facing this research is to promote rapid osteointegration of titanium implants and to minimise the risks of infections by the functionalization with different agents, each designed for a specific action. A patented process gives a multifunctional titanium surface. Introduction. A patented process of surface modification is described. It gives a multifunctional surface with a multiscale roughness (micro and nano topography), that is excellent for osteoblast adhesion and differentiation. It has a high degree of hydroxylation, that is relevant for inorganic bioactivity (apatite-HA precipitation) and it is ready for a functionalization with biological factors. A direct grafting of ALP has been obtained. Moreover, the growth of an antibacterial agent within the surface oxide layer can be useful in order to combine the
Summary. Properties of human amniotic membrane are particularly interesting. To use it as an Advanced Therapeutic Medicinal Product in bone surgery, we are evaluating its association with a potentially osteoinductive scaffold. Introduction. The human Amniotic Membrane (hAM) is known to have a good potential to help the regeneration of tissues. It has been used for 100 years in many medical disciplines because of its properties: a membrane containing stem cells and growth factors, with low immunogenicity and anti-microbial, anti-inflammatory, anti-fibrotic and analgesic properties. Moreover, previous published data showed the possibility of in vitro osteodifferenciation of the whole tissue. We aim to use hAM as an Advanced Therapeutic Medicinal Product for bone repair to treat large defects or pseudarthrosis. So we are studying the association of hAM with nanofiber jet sprayed polycaprolactone (PCL) scaffolds and the possibility to induce its osteodifferenciation. Materials and Methods. HAM from cesarean delivery were provided by a local bank of tissue. A biodegradable microfiber PCL scaffold (∼500 μm thick) was produced using a novel jet spraying technique and provided by Biomedical Tissues society (Nantes, France). We cultured hAM in contact with PCL scaffolds either in MSCs expansion medium or in MSCs osteogenic medium. Then we grafted these montages in an ectopic murin model (in subcutaneous implantation) and we explanted grafted tissues after 1, 2, 4 and 8 weeks. Osteogenic potential was evaluated by immunological studies immediately after in vitro cell studies and after explantations from the mice. Control studies were performed with human mesenchymal stromal cells (hMSCs) seeded on PCL scaffold in MSCs osteogenic medium and cultured with/ without dynamic culture, via an orbital rotator at 150 rpm for 28 days. Cell viability, proliferation and osteoblastic differentiation were evaluated at different times of culture until 28 days. Results. To date, in vitro studies showed a macroscopic mineralization of hAM cultured in MSCs osteogenic medium. Osteogenic potential evaluated by immunological investigations are currently underway. Control studies showed that:. - PCL jet sprayed scaffolds supported proliferation and maintained viability of hMSCs. - Scanning electron microscopy analysis and confocal imaging showed cell attachment with a spread cell morphology after just 1.5 hours. Cells attached along the PCL nanofibres.?. - Entire scaffold depth was infiltrated with cells at days 7 and 28, as seen by DAPI and hematoxylin and eosin staining. - Minimal collagen deposition was evident after 7 days but was observed in significant amounts after 28 days. Static conditions had the greatest collagen matrix production. - Alkaline phosphatse gene expression increased in dynamic conditions compared to 2D cultures. Osteocalcin gene expression increased on PCL scaffolds compared to 2D plastic. - Dynamic loading did not appear necessary for proliferation, infiltration or collagen production. Discussion. The association of the hAM with an osteoinductive scaffold could have 2 benefits:. - The handling of the hAM. - The osteodifferentiation of the hAM without the employment of chemical products (osteoinductive supplements that would need to be use with a “good manufacturing products” validation). These preliminary data showed that PCL scaffolds presented
To explore the therapeutic potential of combining bone marrow-derived mesenchymal stem cells (BM-MSCs) and hydroxyapatite (HA) granules to treat nonunion of the long bone. Ten patients with an atrophic nonunion of a long bone fracture were selectively divided into two groups. Five subjects in the treatment group were treated with the combination of 15 million autologous BM-MSCs, 5g/cm3 (HA) granules and internal fixation. Control subjects were treated with iliac crest autograft, 5g/cm3 HA granules and internal fixation. The outcomes measured were post-operative pain (visual analogue scale), level of functionality (LEFS and DASH), and radiograph assessment.Objectives
Methods
We have developed an animal model to examine the formation of heterotopic ossification using standardised muscular damage and implantation of a beta-tricalcium phosphate block into a hip capsulotomy wound in Wistar rats. The aim was to investigate how cells originating from drilled femoral canals and damaged muscles influence the formation of heterotopic bone. The femoral canal was either drilled or left untouched and a tricalcium phosphate block, immersed either in saline or a rhBMP-2 solution, was implanted. These implants were removed at three and 21 days after the operation and examined histologically, histomorphometrically and immunohistochemically. Bone formation was seen in all implants in rhBMP-2-immersed, whereas in those immersed in saline the process was minimal, irrespective of drilling of the femoral canals. Bone mineralisation was somewhat greater in the absence of drilling with a mean mineralised volume to mean total volume of 18.2% ( Our findings suggest that osteoinductive signalling is an early event in the formation of ectopic bone. If applicable to man the results indicate that careful tissue handling is more important than the prevention of the dissemination of bone cells in order to avoid heterotopic ossification.
Impacted bone allograft is often used in revision joint replacement. Hydroxyapatite granules have been suggested as a substitute or to enhance morcellised bone allograft. We hypothesised that adding osteogenic protein-1 to a composite of bone allograft and non-resorbable hydroxyapatite granules (ProOsteon) would improve the incorporation of bone and implant fixation. We also compared the response to using ProOsteon alone against bone allograft used in isolation. We implanted two non-weight-bearing hydroxyapatite-coated implants into each proximal humerus of six dogs, with each implant surrounded by a concentric 3 mm gap. These gaps were randomly allocated to four different procedures in each dog: 1) bone allograft used on its own; 2) ProOsteon used on its own; 3) allograft and ProOsteon used together; or 4) allograft and ProOsteon with the addition of osteogenic protein-1. After three weeks osteogenic protein-1 increased bone formation and the energy absorption of implants grafted with allograft and ProOsteon. A composite of allograft, ProOsteon and osteogenic protein-1 was comparable, but not superior to, allograft used on its own. ProOsteon alone cannot be recommended as a substitute for allograft around non-cemented implants, but should be used to extend the volume of the graft, preferably with the addition of a growth factor.