Monika Herten

Bridging the gap: bone marrow aspiration concentrate reduces autologous bone grafting in osseous defects.

Although autologous bone grafting represents an effective tool to induce osteogenic regeneration in local bone defects or pseudarthroses, it is associated with significant donor site morbidity and limited by the amount available for grafting. We investigate the potency of bone marrow aspiration concentrate (BMAC) to augment bone grafting and support bone healing. The functional and radiographic outcome of 39 patients with volumetric bone deficiencies treated with BMAC are presented and evaluated in a prospective clinical trial. A collagen sponge (Col) served as scaffold in 12 patients and a bovine hydroxyapatite (HA) was applied in the other 27 individuals. The minimal follow‐up was 6 months. Clinical and radiographic findings were completed by in vitro data. All patients showed new bone formation in radiographs during follow‐up. However, two patients underwent revision surgery due to a lack in bone healing. In contrast to the Col group, the postoperative bone formation appeared earlier in the HA group (HA group: 6.8 weeks vs. Col group 13.6 weeks). Complete bone healing was achieved in the HA group after 17.3 weeks compared to 22.4 weeks in the Col group. The average concentration factor of BMAC was 5.2 (SD 1.3). Flow cytometry confirmed the mesenchymal nature of the cells. Cells from BMAC created earlier and larger colonies of forming units fibroblasts (CFU‐F) compared to cells from bone marrow aspirate. BMAC combined with HA can reduce the time needed for healing of bone defects when compared to BMAC in combination with collagen sponge.

Immunohistochemical characterization of guided bone regeneration at a dehiscence‐type defect using different barrier membranes: an experimental study in dogs

OBJECTIVES The aim of the present study was to evaluate immunohistochemically the pattern of guided bone regeneration (GBR) using different types of barrier membranes. MATERIAL AND METHODS Standardized buccal dehiscence defects were surgically created following implant bed preparation in 12 beagle dogs. Defects were randomly assigned to six different GBR procedures: a collagen-coated bone grafting material (BOC) in combination with either a native, three cross-linked, a titanium-reinforced collagen membrane, or expanded polytetrafluorethylene (ePTFE), or BOC alone. After 1, 2, 4, 6, 9, and 12 weeks of submerged healing, dissected blocks were processed for immunohistochemical (osteocalcin - OC, transglutaminase II - angiogenesis) and histomorphometrical analysis [e.g., bone-to-implant contact (BIC), area of new bone fill (BF)]. RESULTS In general, angiogenesis, OC antigen reactivity, and new bone formation mainly arose from open bone marrow spaces at the bottom of the defect and invaded the dehiscence areas along the implant surface and BOC. At 4 weeks, membranes supporting an early transmembraneous angiogenesis also exhibited some localized peripheral areas of new bone formation. However, significantly increasing BIC and BF values over time were observed in all groups. Membrane exposure after 10-12 weeks was associated with a loss of the supporting alveolar bone in the ePTFE group. CONCLUSION Within the limits of the present study, it was concluded that (i) angiogenesis plays a crucial role in GBR and (ii) all membranes investigated supported bone regeneration on an equivalent level.

Lateral ridge augmentation using particulated or block bone substitutes biocoated with rhGDF-5 and rhBMP-2: an immunohistochemical study in dogs.

OBJECTIVES The aim of the present study was to immunohistochemically evaluate lateral ridge augmentation using a particulated (BOG) or block (BOB) natural bone mineral biocoated with rhGDF-5 and rhBMP-2 in dogs. MATERIALS AND METHODS Three standardized box-shaped defects were surgically created at the buccal aspect of the alveolar ridge in each quadrant of eight beagle dogs. After 2 months of healing, the chronic-type defects were randomly allocated in a split-mouth design to either (i) BOG or (ii) BOB biocoated with (a) rhGDF-5 or (b) rhBMP-2, respectively. Uncoated grafts served as controls. After 3 and 8 weeks, dissected blocks were prepared for immunohistochemical [osteocalcin (OC)] and histomorphometrical analysis [e.g. area (mm(2)) of new bone fill (BF), newly formed mineralized (MT) and non-mineralized tissue (NMT)]. RESULTS rhBMP-2 biocoated BOG revealed significantly highest BF and MT values at 3 (upper and lower jaws - UJ/LJ - compared with BOG) and 8 weeks (UJ - compared with rhGDF-5). Biocoating of BOB using both rhGDF-5 and rhBMP-2 resulted in significantly increased MT values at 8 weeks (UJ/LJ - compared with BOB). In all groups, NMT adjacent to BOG and BOB scaffolds revealed pronounced signs of an OC antigen reactivity. CONCLUSIONS Within the limits of the present study, it was concluded that both rhGDF-5 and rhBMP-2 have shown efficacy; however, their bone regenerative effect was markedly influenced by the carrier.

Combined use of platelet-rich plasma and autologous bone grafts in the treatment of long bone defects in mini-pigs

The use of platelet-rich plasma (PRP) for improving of bone defect healing is discussed controversially. The aim of this study was to assess the effect of PRP in combination with autologous cancellous graft on bone defect healing in a critical metaphyseal long bone defect. A critical size defect in the tibial metaphysis of 16 mini-pigs was filled either with autologous cancellous graft as control group or with autologous cancellous graft combined with autologous PRP. Compared to native blood platelets were enriched about 4.9-fold in the PRP. After 6 weeks, the specimens were assessed by X-ray and histological evaluation. Histomorphometrical analysis revealed that the area of new bone was significantly higher in the PRP group concerning the central area of the defect zone (p<0.02) as well as the cortical defect zone (p<0.01). All defects showed substantial new bone formation, but only defects of the PRP group regenerated entirely. The PRP group was superior to the control group even in the semi-quantitative assessment of the osseous bridging in both observed areas of the defect. Within the limits of the present study it could be demonstrated that PRP combined with autologous cancellous graft leads to a significantly better bone regeneration compared to isolated application of autologous cancellous graft in an in vivo critical size defect on load-bearing long bones of mini-pigs.

Biodegradation of different synthetic hydrogels made of polyethylene glycol hydrogel/RGD-peptide modifications: an immunohistochemical study in rats

AIM The aim of the present study was to investigate the pattern of biodegradation of different polyethylene glycol (PEG) hydrogel/RGD-peptide modifications in rats. MATERIAL AND METHODS Two different hydrogels were employed: (i) a combination of four-arm PEG-thiol, M(n)=2.3 kDa, and eight-arm PEG-acrylate, M(n)=2.3 kDa (PEG1); and (ii) a combination of four-arm PEG-thiol, M(n)=2.3 kDa, and four-arm PEG-acrylate, M(n)=15 kDa (PEG2). Both PEG1 and PEG2 were either used alone or combined with a nine amino acid cys-RGD peptide (RGD). A non-cross-linked porcine type I and III collagen membrane [BioGide (BG)] served as control. Specimens were randomly allocated in unconnected subcutaneous pouches separated surgically on the back of 60 wistar rats, which were divided into six groups (1, 2, 4, 8, 16, and 24 weeks). Specimens were prepared for histological (tissue integration, foreign body reactions, biodegradation) and immunohistochemical (angiogenesis) analysis. RESULTS All materials investigated revealed unimpeded and comparable tissue integration without any signs of foreign body reactions. While BG exhibited transmembraneous blood vessel formation at 1 week, all PEG specimens were just surrounded by a well-vascularized connective tissue. The hydrolytic disruption of PEG1 and PEG1/RGD specimens was associated with an ingrowth of blood vessels at 4 weeks. Biodegradation times were highest for PEG1 (24 weeks)>PEG1/RGD (16 weeks)>BG (4 weeks)>PEG2=PEG2/RGD (2 weeks). CONCLUSION Within the limits of the present study, it was concluded that (i) all materials investigated revealed a high biocompatibility and tissue integration, and (ii) hydrogel biodegradation was dependent on PEG composition.

Platelet-Rich Plasma on Calcium Phosphate Granules Promotes Metaphyseal Bone Healing in Mini-Pigs

The role of platelet‐rich plasma (PRP) as a promoter of bone healing remains controversial. The aim of this study was to investigate the effect of PRP in combination with calcium phosphate granules (CPG) on bone defect healing in a metaphyseal long bone defect. A metaphyseal bone defect at the proximal tibia of 16 mini‐pigs was filled with CPG combined with autologous PRP or CPG solely (control group). The PRP showed 4.4‐fold more platelets compared to peripheral blood. Six weeks after surgery the radiological and histomorphometrical evaluations showed significantly more bone formation in the PRP group in the central area of the defect zone (p < 0.01) as well as the cortical defect zone (p < 0.04). Furthermore, the resorption rate of CPG was increased in animals who received PRP. Nevertheless there were only isolated instances of complete osseous bridging of the bone defects even in the PRP group. This study demonstrates that a PRP‐CPG composit promotes bone regeneration but does not lead to a solid fusion of a tibial defect in mini‐pigs.

Cell therapy in bone healing disorders

In addition to osteosynthetic stabilizing techniques and autologous bone transplantations, so-called orthobiologics play an increasing role in the treatment of bone healing disorders. Besides the use of various growth factors, more and more new data suggest that cell-based therapies promote local bone regeneration. For ethical and biological reasons, clinical application of progenitor cells on the musculoskeletal system is limited to autologous, postpartum stem cells. Intraoperative one-step treatment with autologous progenitor cells, in particular, delivered promising results in preliminary clinical studies. This article provides an overview of the rationale for, and characteristics of the clinical application of cell-based therapy to treat osseous defects based on a review of existing literature and our own experience with more than 100 patients. Most clinical trials report successful bone regeneration after the application of mixed cell populations from bone marrow. The autologous application of human bone marrow cells which are not expanded ex vivo has medico-legal advantages. However, there is a lack of prospective randomized studies including controls for cell therapy for bone defects. Autologous bone marrow cell therapy seems to be a promising treatment option which may reduce the amount of bone grafting in future.

Dimensional ridge alterations following socket preservation using a nanocrystalline hydroxyapatite paste: a histomorphometrical study in dogs.

The aim of the study was to evaluate the histological response and dimensional ridge alterations following application of a nanocrystalline hydroxyapatite paste (NHA) into fresh extraction sockets in dogs. Immediately following vertical tooth separation and extraction, NHA was inserted in the extraction socket of the second molar in the lower jaws of 10 dogs. Untreated extraction sites on the opposite side served as controls. Wounds were closed using resorbable sutures after vertical flap elevation. After three and six months, 5 animals were killed. Lingual and buccal bone height, alveolar wall and total bone width 1, 3 and 5mm underneath the top of the crest were evaluated. Histological analysis revealed a high variability of NHA resorption and osteoconductive properties with different rates of material resorption. No statistically significant differences could be observed between the corresponding aspects of test and control sites. Both groups revealed higher alveolar wall resorption on the buccal than on the lingual side at both time periods. NHA does not seem to be useful for socket preservation procedures since it failed to prevent dimensional ridge alterations while revealing osseous integration but unpredictable material resorption. The role of non-resorbed hydroxyapatite remnants for implant placement is unclear and requires further investigation.

Bone Marrow Aspiration Concentrate and Platelet Rich Plasma for Osteochondral Repair in a Porcine Osteochondral Defect Model

Background Bone marrow aspiration concentrate (BMAC) may possess a high potency for cartilage and osseous defect healing because it contains stem cells and multiple growth factors. Alternatively, platelet rich plasma (PRP), which contains a cocktail of multiple growth factors released from enriched activated thrombocytes may potentially stimulate the mesenchymal stem cells (MSCs) in bone marrow to proliferate and differentiate. Methods A critical size osteochondral defect (10×6 mm) in both medial femoral condyles was created in 14 Goettinger mini-pigs. All animals were randomized into the following four groups: biphasic scaffold alone (TRUFIT BGS, Smith & Nephew, USA), scaffold with PRP, scaffold with BMAC and scaffold in combination with BMAC and PRP. After 26 weeks all animals were euthanized and histological slides were cut, stained and evaluated using a histological score and immunohistochemistry. Results The thrombocyte number was significantly increased (p = 0.049) in PRP compared to whole blood. In addition the concentration of the measured growth factors in PRP such as BMP-2, BMP-7, VEGF, TGF-β1 and PDGF were significantly increased when compared to whole blood (p<0.05). In the defects of the therapy groups areas of chondrogenic tissue were present, which stained blue with toluidine blue and positively for collagen type II. Adding BMAC or PRP in a biphasic scaffold led to a significant improvement of the histological score compared to the control group, but the combination of BMAC and PRP did not further enhance the histological score. Conclusions The clinical application of BMAC or PRP in osteochondral defect healing is attractive because of their autologous origin and cost-effectiveness. Adding either PRP or BMAC to a biphasic scaffold led to a significantly better healing of osteochondral defects compared with the control group. However, the combination of both therapies did not further enhance healing.

Glycosylation processing inhibition by castanospermine prevents experimental autoimmune encephalomyelitis by interference with IL-2 receptor signal transduction

In this study, we explored the therapeutic targets of the glycosylation processing inhibitor, castanospermine (CAST), in murine passive transfer experimental autoimmune encephalomyelitis (EAE), a model disease of multiple sclerosis. By using lymphocytic-endothelial adhesion and transmigration assays, FACScan and Western blotting, we defined the effects of CAST on expression, function and signal transduction of glycoproteins crucial in the pathophysiology of this disease. CAST prevented clinical signs of EAE and completely inhibited inflammatory CNS infiltrates associated with this disease. Here, we showed that CAST blocks antigen-induced lymphocytic activation and clonal expansion in a dose-dependent manner. Importantly, we observed that CAST strongly impairs IL-2-induced signal transduction of the IL-2 receptor. In contrast, neither expression nor binding ability of the IL-2 receptor was affected by this drug. In addition, we were able to exclude major effects of CAST on expression and function of different glycoproteins important in antigen presentation as well as lymphocytic-endothelial adhesion and transmigration. In conclusion, CAST strongly interferes in the signal transduction of the IL-2 receptor. This could explain both inhibitory effects of CAST in clonal T cell expansion and development of transfer EAE. This relatively selective pharmacological effect of CAST highlights its potential as a novel immunomodulatory approach in multiple sclerosis.