Objectives

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.

INTRODUCTION. Autologous bone grafts are considered gold standard in the repair of bone defects. However they are limited in supply and are associated with donor site morbidity. This has led to the development of synthetic bone graft substitute (BGS) materials, many of which have been reported as being osteoinductive. The structure of the BGS is important and bone formation has been observed in scaffolds with a macroporous morphology. Smaller pores termed ‘strut porosity’ may also be important for osteoinduction. The aim of this study was to compare the osteoinductive ability of one silicate-substituted calcium phosphate (SiCaP) with differing strut porosities in an ectopic ovine model. Our hypothesis was that SiCaP with greater strut porosity would be more osteoinductive. METHODS. The osteoinduction of SiCaP BGS with two different strut porosities (AF and AF++) was investigated. The materials had an identical chemical composition and morphological structure but differing strut porosity (AF=22.5%, AF++=47%). Implants were inserted into the paraspinal muscles in skeletally mature sheep. Procedures were carried out in compliance with UK Home Office regulations. There were 12 implants in each group. Implants remained in vivo for 8 and 12 weeks and on retrieval were prepared for undecalcified histology. Sections were stained and examined using light microscopy. A line intersection method was used to quantify bone, implant and implant surface/bone contact within seven random regions of interest along each implant. A Mann-Whitney U test was used for statistical analysis where p values < 0.05 were considered significant. RESULTS. Bone formation was observed to be greater in the AF++ group at 8 (AF=0.2%+/−0.15; AF++=0.44%+/−0.12) and significantly higher at 12 weeks (AF=1.33% +/−0.84; AF++=6.17%+/−1.51) (p=0.04). Significantly higher implant surface/bone contact was observed in the AF++ group at 8 (AF=0.67%+/−0.52; AF++=3.30%+/−1.17) (p=0.04) and 12 weeks (AF=3.06%+/−1.89; AF++=21.82%+/−5.59) (p=0.01). The % implant measured was less in the AF++ group at 8 (AF=39.06%+/−1.26; AF++=33.09%+/−2.14) and 12 weeks (AF=36.05% +/−3.55; AF++=30.60%+/−2.29) but this was not significant. Histology revealed bone formation within BGS strut pores measuring < 50um. Endochondral and intramembranous ossification were also observed in both groups. DISCUSSION. The results indicate that higher strut porosity promotes greater osteoinduction in SiCaP materials. This could be attributed to the micropores providing a greater surface area for the action of growth factors and osteoblasts leading to the formation of bone at an earlier time point. Endochondral ossification was an unusual finding as this is usually associated with bone formation secondary to Bone Morphogenetic proteins (BMPs). This suggests that the osteoinductive mechanisms by SiCaP may involve cytokines such as BMPs

Aims. Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs). Methods. EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron microscopy, nanoparticle tracking analysis, and the expression of CD9 and CD63 markers by western blot. The effect of the obtained EVs on osteoinduction was evaluated in vitro on human umbilical cord MSCs by messenger RNA (mRNA) expression analysis of bone markers, alkaline phosphatase activity (ALP), and calcium (Ca. 2+. ) content. Results. Osteogenic differentiation of MSCs was confirmed when treated with UC-isolated EVs. In order to disprove that the effect was due to co-isolated proteins, EVs were isolated by SEC. Purer EVs were obtained and proved to maintain the differentiation effect on MSCs and showed a dose-dependent response. Conclusion. PL-derived EVs present an osteogenic capability comparable to PL treatments, emerging as an alternative able to overcome PL and PRP limitations. Cite this article: Bone Joint Res 2020;9(10):667–674

Unicameral bone cyst is a frequent benign lesion of the child’s skeleton of unclear ethopathology and scarce symptoms.Possible complications and limits in the child’s activities necessitate an active approach to management. Intracystic injection of corticosteroid depot, curettage of the cyst and bone grafting and osteoinduction procedures are three frequently used therapeutic procedures.Having in mind that attitudes regarding initial treatment are still heterogeneous, in period 1992–2001, we have conducted a study aimed at making a comparative analysis of those commonly applied methods of treatment. The prospective randomised study included 37 patients treated with corticosteroid injections, 32 patients treated with curettage and bone grafting and 31 patients treated with osteoinduction procedure.As osteoinductive material we used mixture of demineralised bone matrix powder and autologous bone marrow. Results evaluation was based on criteria designed by Neer,Campannaci and Capanna.Mean follow up period was 6,5 years. In our patients results of initial treatment were good, regardless of the applied method and they do not significantly differ from the results of other authors.No significant difference in treatment outcome was statistically revealed and frequency of recurrence does not vary in relation to applied method. Mean failure rate was 18%,mean recurrence rate 10% and mean rate of usual complications 17%. Under conditions of similar efficiacy, it is justified to apply a less invasive method in the initial treatment. Methods of corticosteroid injection and osteoinduction are advantageous in cysts with physeal and epiphyseal propagation especially in the upper limb. Besides, those methods are characterised by simplicity, safety and short duration of intervention and hospitalisation and short limitation of the child’s activity.In exceptionally large cysts, as well as in cysts localised in the femoral neck and complicated with displaced fracture which requires reposition and osteosynthesis, the advantage is given to curetage and bone grafting

Purpose: Interbody fusion cages have met with great success as an adjunct in the treatment of painful degenerative disc disease. One of the limitations is the need for the use of autogenous cancellous bone graft. In preclinical studies recombinant human bone morphogenetic protein-2 (rhBMP-2) delivered in a variety of carriers has been shown to be an effective substitute for autogenous bone, resulting in more rapid and reliable healing than that seen in control groups. The goal of this study was to report the early results of the first human trial attempting to use rhBMP-2 in interbody fusion cages. Methods: This study was an FDA approved IDE multicenter pilot study. From 1/97 to 4/97, 14 patients were entered into a prospective, randomized trial. All patients had single level lumbar degenerative disc disease that was refractory to prolonged nonoperative care and were candidates for anterior interbody fusion of L4-5 or L5-S1. After consent, patients were randomized to either the control group (N-3) and received autogenous bone inside tapered titanium fusion cages (NOVUS LT, Sofamor Danek Memphis, TN) or to the investigational group (N = 11) and received rhBMP-2 (1.5 mg/ml)(Genetics Institute, Cambridge MA) delivered in a collagen sponge (Helistat, Integra Life Sciences, Plainsboro, NJ) inside the fusion cages. Depending on the size, the sponge in each cage was soaked with from 1.3 to 2.6 ml of the rhBMP2 solution. Patients were followed at regular intervals with plain x-ray, CT scan with reconstruction, and a full panel of blood tests. Radiographs were reviewed by an independent blinded radiologist with fusion defined as < 5 degrees of sagittal motion, absence of radiolucent lines, and presence of continuous bone through the cages. Clinical results were assessed using an outcomes questionnaire including the SF-36 general health status and Oswestry low back specific instruments. Results: All 14 patients were available for 1-year follow-up. No cages displaced and no further surgeries were required. Mean hospital stay was 2.0 days for the rhBMP-2 patients compared to 3.3 days for the autograft controls. Of the 11 rhBMP2 patients, 10 of 11 were judged to be fused at 3 months. At 6 months and 1-year all 11 rhBMP-2 patients were noted to have a solid arthrodesis. Of the 3 control patients, 2 had solid arthodesis and one had an apparent nonunion at 1 year. On sagittal CT scan reconstruction new bone growth was seen throughout and anterior to the cages that were filled with rhBMP-2. No patients had bone formation outside of the desired area. The control patient with the nonunion had a halo surrounding the cage on the sagittal CT reconstruction. This patient had persistence of low back pain. Compared to preop, the Oswestry scores at 3 months were decreased in the rhBMP-2 group (39 to 30) compared to controls which were increased (35 to 43) and both mean scores were similar at 6 months (28 and 27). Conclusion: The preliminary results from this clinical trial with rhBMP-2 inside interbody fusion cages were excellent and support a larger pivotal trial. The arthrodesis was found to occur more rapidly and reliably than in the controls, although the sample size was limited. In addition to faster bone healing, a major advantage was the elimination of bone graft donor site morbidity and realization of decreased hospital stay. No evidence of excessive bone formation or systemic complications occurred. Moreover, this study provides one of the first demonstrations of consistent and unequivocal osteoinduction by a recombinant growth factor in humans

The use of autologous grafts for vertebral arthrodesis is associated with a number of complications that should be properly considered: pain at the harvesting site, increased blood loss, prolonged surgical time, and additional scar. Moreover, in many cases, the amount of autologous bone is insufficient. Novel materials, either natural or synthetic, are therefore needed to be used as bone substitutes in vertebral surgery. For this purpose, a number of synthetic materials have been developed, their characteristics varying considerably in terms of ostoinduction, osteoconduction, biomecanics, and cost.

In particular, clinical and experimental studies have highlighted the potential of demineralized bone matrix (DBM), alone or in combination with autologous grafts, and of collagenic mineralized matrix (Healos).

Aim of this study was the evaluation of the clinical value of these materials in vertebral surgery. We have analyzed a series of 60 patients who underwent vertebral arthrodesis by the addition of either DBM (30 cases) or Healos (30 cases).

Bone substitutes were used both in posterior-lateral arthrodeses (on one side, the other being treated with autologous graft as a control) and in intersomatic arthrodeses in association with titanium or carbon fiber scaffolds.

Patients were followed-up for a minimum 1-year interval, and evaluated with regard to clinical (Oswestry score, SF-36) and radiographic (static and dynamic X-rays, spiral CT, MRI) parameters. The area of arthrodesis was independently analyzed by three independent observers.

Clinical results showed the reliability of both materials as a tool for a stable arthrodesis, since they were found to be able to achieve results comparable to those obtained with autologous grafts in the control arm of the study.

Numerous growth factors, together with bone morphogenetic proteins (BMP), are involved in the biological mechanism of bone induction. BMP 7 combined with collagen type 1 has shown to be effective in triggering the osteoinductive mechanism in the presence of bone defects as an alternative to bone graft. The aim of the present study is to present the experience gained at the Orthopaedic Clinic of Catania with BMP in lower limb non-union or pseudoarthrosis with bone defect after skeletal stabilisation.

From December 2003 to May 2004 at the Orthopaedic Clinic Of Catania four patients were treated using OP 1. The study group comprised three women and one man with a mean age at the time of operation of 27 years (range 23– 32 years). One subject presented with non-union, two patients with normotrophic pseudoarthrosis and one with atrophic pseudoarthrosis. In all cases the bone defect was localised at the tibia. All patients were treated surgically with skeletal stabilisation (one with a monolateral external fixator, one with a T2 intramedullary nail and two with a Gross-Kempf nail) always after decortication of Judet.

Three patients healed with a mean follow-up of 5 months. One subject is still under treatment. None of the patients have any pain and demonstrate complete weight bearing and functional capacity. X-rays showed good consolidation of bone defect.

Clinical and radiographic results obtained at this point are more than fair. Healing time was short without any side effects. It is important to verify whether, in the treatment of lower limb non-union and or pseudoarthrosis, the osteoinductive capacity of the OP 1 could provide the same valid results without good reduction and skeletal stabilisation. The use of OP 1 is extremely interesting for many reasons. However, it would be extremely important to test whether the biological effect is not exclusively related to an optimal mechanical stability.

Introduction

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.

Objectives

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.

There are no efficient treatment options for osteoarthritis (OA) that delay further progression. Besides osteoinduction, there is growing evidence of also anti-inflammatory, angiogenetic and neuroprotective effects of biodegradable magnesium-based biomaterials. Their use for the treatment of cartilage lesions in contrast is not well-evaluated yet. Mg-cylinders were analysed in an in vitro and in vivo OA model. In vitro, SCP-1 stem cell line was analysed under inflammatory conditions and Mg-impact. In vivo, small Mg- and WE43 alloy-cylinders (1mm × 0,5mm) were implanted into the subchondral bone of the knee joint of 24 NZW rabbits after establishment of OA. As control, another 12 rabbits received only drill-holes. µCT-scan were performed and assessed for changes in bone volume and density. After euthanasia, cartilage was evaluated macroscopically and histologically after Safranin-O-staining. Furthermore, staining with CD271 directed antibody was performed to assess neuro-reactivity. In vitro, an increased gene expression of extracellular matrix proteins as collagen II or aggrecan even under inflammatory conditions was observed under Mg-impact. In vivo, µCT evaluation revealed twice-elevated values for bone volume in femoral condyles with Mg-cylinders compared to controls while density remained unchanged. Cartilage showed no significant differences between the groups. Mg- and WE-samples showed significantly lower levels of CD271+ cells in the cartilage and bone of the operated joints than in non-operated joints, which was not the case in the Drilling-group. Furthermore, bone in operated knees of Drilling-group showed a strong trend to an increase in CD271+ cells compared to both Cylinder-groups. Counting of CD271+ vessels revealed that this difference was attributable to a higher amount of these vessels. The in vitro results indicate a potential cartilage regenerative activity of the degradable Mg-based material. While so far there was no positive effect on the cartilage itself in vivo, implantation of Mg-cylinders seemed to reduce pain-mediating vessels. Acknowledgements: This work is funded by the German Research Foundation (DFG, project number 404534760). We thank Björn Wiese for production of the cylinders

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 osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

Aim. Periprosthetic joint infections follow 1-3% of arthroplasty surgeries, with the biofilm nature of these infections presenting a significant treatment challenge. 1. Prevention strategies include antibiotic-loaded bone cement; however, increases in cementless procedures means there is an urgent need for alternative local antimicrobial delivery methods. 2. A novel, ultrathin, silica-based sol-gel technology is evaluated in this research as an anti-infective coating for orthopaedic prosthetic devices, providing local antibiotic release following surgery. Method. Reduction in clinically relevant microbial activity and biofilm reduction by antimicrobial sol-gel coatings, containing a selection of antibiotics, were assessed via disc diffusion and microdilution culture assays using the Calgary biofilm device. 3. Proliferation, morphology, collagen, and calcium production by primary bovine osteoblasts cultured upon antibiotic sol-gel surfaces were examined, and cytotoxicity evaluated using Alamar blue staining and lactate dehydrogenase assays. Concentrations of silica, calcium and phosphorus compounds within the cell layer cultured on sol-gel coatings and concentrations eluted into media, were quantified using ICP-OES. Furthermore, cellular phenotype was assessed using alkaline phosphatase activity with time in culture. Results. Low antibiotic concentrations within sol-gel had an inhibitory effect on clinically relevant biofilm growth, for example 0.8 mg ml. -1. tobramycin inhibited clinically isolated S. aureus (MRSA) growth with an 8-log reduction in viable colony forming units. There was no significant difference in metabolic activity between untreated and sol-gel exposed primary bovine osteoblasts in elution-based assays. Reduction (2-fold) in metabolic activity in direct contact assays after 48 hours exposure was likely to be due to increased osteoinduction, whereas no impact upon cell proliferation were observed (p=0.92 at 14 days culture). The morphology of primary osteoblasts was unaffected by culture on sol-gel coatings and collagen production was maintained. Calcium containing nodule production within bovine osteoblastic cells was increased 16-fold after 14 days culture upon sol-gel. Conclusions. The ultrathin sol-gel coating showed low cytotoxicity, strong biofilm reducing activity and antimicrobial activity, which was comparable to antibiotics alone, demonstrating that sol-gel delivery of antibiotics could provide local antimicrobial effects to inhibit PJI growth without the need for bone cement. Future work will develop and evaluate sol-gel performance in an ex vivo explant bone infection model which will reduce the need for animal experimentation

Objectives. Despite promising results have shown by osteogenic cell-based demineralized bone matrix composites, they need to be optimized for grafts that act as structural frameworks in load-bearing defects. The aims of this study is attempt to assess the effects of laser perforations on osteoinduction in cortical bone allografts. Methods. Sixteen wistar rats were divided into two groups according to the type of structural bone allograft; the first: partially demineralized only (Donly) and the second: partially demineralized laser-perforated (DLP). Trans-cortical holes were achieved by Er:YAG laser at a wave length of 2.94 µm in four rows of three holes approximated cylindrical holes 0.5 mm in diameter, with centres 2.5 mm apart. Histologic and histomorphometric analysis were performed at 12 weeks. Results. Statistical analysis showed significant differences between the 2 groups at 3 months. Results showed that partially demineralized laser-perforated grafts had substantially higher incorporation by woven bone than partially demineralized grafts; yet this difference at the interface gap remained insignificant. In DLP allografts healing at the junction was more complete and a wider area was in contact with host and graft surfaces. Conclusions. Based on the results of this study, it may be concluded that surface changes induced by Er:YAG laser, accelerated bone healing with good osteoinduction results and the process might have improved, if they could have been supplemented with the proper stipulations

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, osteoinduction, and angiogenesis in hMSCs and HUVECs were enhanced by the supernatant. Conclusions. The current study demonstrates the potential of a new point-of-care strategy for regenerative medicine using skeletal muscle supernatant. This attractive approach and readily-available material could be a promising option for tissue repair/regeneration in the clinical setting. Cite this article: M. Yoshikawa, T. Nakasa, M. Ishikawa, N. Adachi, M. Ochi. Evaluation of autologous skeletal muscle-derived factors for regenerative medicine applications. Bone Joint Res 2017;6:277–283. DOI: 10.1302/2046-3758.65.BJR-2016-0187.R1

It is very important to fix implant to bone. Bioactive materials as hydroxyapatite or glass-ceramics have bone-bonding ability. Hydroxyapatite-coating is applied to cementless THA or TKA. I and coworkers investigated bone-bonding mechanism of bioactive material and found that bone-like apatite formation play key role for bonding. If the surface of metal is changed to form apatite on it in body, the inert metal changes into bone-bonding material. We developed alkaline and heat treatment of titanium to change titanium to bone –bonding material as follows. At first, titanium is dipped in 5N NaOH solution for 24 hours, at second the metal is washed in pure water and finally it is sintered in 500 degree C for 2 hours. The treated surface has bioactivity, bone bonding ability like hydroxyapatite. The advantage of this treatment over hydroxyapatite-coating procedure is to treat the porous surface without any change of pore figures. As to hydroxyapatite-coating procedure, pore of the small diameter is filled with hydroxyapatite and pore figures are change. We applied this alkaline and heat treatment to cementless THA and its good results of more than ten years was reported. Porous titanium can be changed to bioactive material by alkaline and heat treatment. This bioactive porous titanium was found to have a property of material-induced osteoinduction, that is, the bone formation in pore of porous titanium implanted in canine back muscle. They can be used for bone substitute for big bone defect. We used two procedures to make porous titanium, sintering of titanium powder with spacer particle of ammonium sulfate and selective lazar melting. The latter procedure can produce any type of pore structure of titanium. Selective laser melting was employed to fabricate porous Ti implants (diameter 3.3 mm, length 15 mm) with a channel structure comprising four longitudinal square channels, representing pores, of different diagonal widths, 500, 600, 900, and 1200 micrometer. These were then subjected to chemical and heat treatments to induce bioactivity. Significant osteoinduction was observed in widths 500 and 600 micrometer, with the highest observed osteoinduction occurring at 5 mm from the end of the implants. A distance of 5 mm probably provides a favorable balance between blood circulation and fluid movement. New bioactive bone cement is another topic of the application of bioactive titanium in this lecture. The bone cement contains barium sulphate for radiocontrast. We developed a procedure to replace barium sulphate with bioactive titanium powder. This new bone cement has not only better biocompatibility than conventional cement but also bone bonding ability. It is potent material for the fixation of implant to bone. I will speak the evaluation of this cement using canine model of THA

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 osteoinduction through the regulation of host cells. In conclusion, our results show a relation between DBM particle size, structural modification of the collagen and in vivo osteoinductivity. The medium particles represent a good compromise between no modification (largest particles) and excessive modification (smallest particles) of collagen structure, yielding highest osteoinduction. We believe that these results can guide researchers to use DBM particles of 0.5–1 mm size range in applications aimed at inducing new bone formation, obtaining results more comparable and reliable among different research groups. Furthermore, we suggest to carefully analyze the structure of the collagen when a collagen-based biomaterial is used alone or in association with cells to induce new bone formation

We report our 4 years’ experience using of demineralized human bone matrix (DBM) in the treatment of complex pathology characterised by bone loss or less regenerating ability, such as congenital or secondary bone mal-union, osteomyelitis, aseptic prosthetic failure, complex bone loss fractures, etc. Considering the known limitations of autologous transplants (limited quantity, infections and fractures of donor sites, operative and bleeding time increase, abdominal herniations, etc.), we have searched in the literature for alternative materials that would be as similar to the osteoconduction and osteoinduction ability of autologous transplant as possible, respecting bio- and immunocompatiblity. Since May 2000 we have used DBM in 50 cases: the first 15 patients with mixed technique (DBM and autologous transplant) and then the other 35 only with DBM. We have controlled each patient clinically and by X-ray: average follow up 34 months. With the same type of pathology and operative technique we have observed a similar recovery with both techniques (DBM with or without autologous transplant); in addition, in patients treated with rigid osteo-synthesis or in patients with osteoporosis we have noted early bone regeneration and no complications with respect to rejection or to osteolysis at the surgical site. In our opinion, this confirms the good osteoconduction and osteoinduction ability of DBM

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 osteoinduction but also by direct bone formation. In vitro, ALLO-P2 overexpressed markers such as ALP compared to BM-MSC isolated from the same donors, suggesting their engagement into the osteogenic lineage. In vivo, a single dose of ALLO-P2 significantly enhanced bone neoformation 14 days post-administration over the calvaria of NMRI-Nude mice compared to the control excipient. Histological analyses and mouse/human type I collagen double-immunolabelling revealed the presence of mineralized bone nodules of mixed host and donor origins in mice administered with ALLO-P2. Together, these results show that ALLO-P2 is a potential promising clinical candidate to promote bone repair, since it can be produced at high yields, is injectable and boosts ossification mechanisms involved in the physiological repair process

A combination of stem cell therapy and tissue engineering is emerging as one of the most promising approaches for skeletal tissue repair and regeneration. Osteoinduction of human bone marrow mesenchymal stem cells (MSCs) is initiated through local signals or growth factors, of which the bone morphogenetic proteins (BMPs) are the best characterised. Cytomodulin-1 (CM-1), a synthetic heptapeptide with functional similarity to members of the TGF-B super family, has been classified as a novel growth factor associated with osteoinduction of MSCs. However, the effects of CM-1 on human bone MSCs are still unclear. The aim of this study was to determine any effects for CM-1 and its scrambled control (CM-1 SCRAM) on the proliferation and differentiation of human bone marrow MSCs along the osteogenic lineage. Primary human bone marrow MSCs were cultured in the presence of CM-1 and CM-1 SCRAM at a range of concentrations (10-8M – 10-6M) in vitro for up to three weeks. 100 ng/mL of recombinant human BMP-2 (rhBMP-2) was used as a positive control. At the end of the culture period, histological and biochemical assays were carried out on the cultures. Biochemical assays revealed that 10-7M of CM-1 significantly stimulated alkaline phosphatase specific activity compared with the negative control group (P< 0.05) in a similar way to the rhBMP-2 positive control group. These data were supported by an observed increase in positive alkaline phosphatase staining in the 10-7M of CM-1 and rhBMP-2 treated cells. However, total DNA content was not significantly different between any of the groups. This study indicated the potential of using CM-1 as an osteogenic growth factor for skeletal tissue regeneration which may provide an alternative approach to meet the major clinical need in orthopaedics and craniofacial surgery. * Cytomodulin-1 and the scrambled control were genuine gifts from Professor (emeritus) Rajendra S. Bhatnagar at the Department of Bioengineering, University California Berkley, USA

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 osteoinduction. Bioactive glasses can be doped with antibacterial ions (silver, copper, etc.) preserving their biocompatibility and bioactivity and, at the same time, acquiring antibacterial properties. Thus, it is possible to produce composite cements that combine the properties of the polymer matrix with those of the inorganic filler, overcoming the main problems associated with the use of antibiotics. An additional possibility is the addition of essential oils, vegetable oils with remarkable antibacterial properties