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Background: The one-step membrane technique, using a human acellular dermal matrix (hADM), is an experimental method for treating large bone defects. This eliminates the need for the Masquelet membrane induction step, shortening the procedure while maintaining effectiveness. However, previous studies showed that colonizing hADM with bone marrow mononuclear cells (BMC) worsens healing, likely due to the presence of CD8+ lymphocytes, which negatively affect bone regeneration. This study aims to investigate whether the negative impact of BMC on bone healing in this technique is due to the CD8+ cell population., Materials and Methods: A 5 mm femoral defect was created in 25 male Sprague-Dawley rats, divided into three groups (G1-G3). BMC were isolated from syngenic donor rats, with CD8+ lymphocytes removed magnetically from the BMC fraction in one group. The defects were filled with bone chips and wrapped with differently treated hADM: G1 received native hADM, G2 received hADM+BMC, and G3 received hADM+BMC-CD8. After 8 weeks, the femurs were evaluated through radiological, biomechanical, and histological examinations., Results: Bone defects and bone mineral density (BMD) were significantly improved in G3 (hADM+BMC-CD8) compared to G2 (hADM+BMC). Bone volume, bone formation, and median bending stiffness were higher in G3. Immunohistological analysis showed a significant decrease in CD8 cell count in G3, with a lower percentage of IFNγ-producing cells compared to G2., Conclusion: Depleting CD8+ cells from BMC before colonizing hADM significantly improved bone healing, likely due to changes in the local mediator environment. This suggests that preoperative colonization with CD8+-depleted BMC could enhance the one-step membrane technique., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Penna-Martinez, Kammerer, Stützle, Fees, Behr, Schaible, Schröder, Verboket, Neijhoft, Marzi, Nau and Henrich.)

Purpose: The Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. Compared to mature periosteum, the induced membrane differs significantly. However, both play a crucial role in bone regeneration. As part of a histological and radiological post-evaluation of an earlier project, we analyzed the influence of the granule size of the bone void filler Herafill ® on development of periosteum regrowth in a critical size defect., Methods: We compared three different sizes of Herafill ® granules (Heraeus Medical GmbH, Wehrheim) in vivo in a rat femoral critical size defect (10 mm) treated with the induced membrane technique. After 8 weeks healing time, femurs were harvested and taken for histological and radiological analysis., Results: A significantly increased regrowth of periosteum into the defect was found when small granules were used. Large granules showed significantly increased occurrence of bone capping. Small granules lead to significant increase in callus formation in the vicinity to the membrane., Conclusion: The size of Herafill ® granules has significant impact on the development of periosteal-like structures around the defect using Masquelet's induced membrane technique. Small granules show significantly increased regrowth of periosteum and improved bone formation adjacent to the induced membrane., (© 2020. The Author(s).)

The Masquelet technique for the treatment of large bone defects is a two-stage procedure based on an induced membrane. The size of a scaffold is reported to be a critical factor for bone healing response. We therefore aimed to investigate the influence of the granule size of a bone graft substitute on bone marrow derived mononuclear cells (BMC) supported bone healing in combination with the induced membrane. We compared three different sizes of Herafill® granules (Heraeus Medical GmbH, Wehrheim) with or without BMC in vivo in a rat femoral critical size defect. A 10 mm defect was made in 126 rats and a membrane induced by a PMMA-spacer. After 3 weeks, the spacer was taken out and membrane filled with different granule sizes. After 8 weeks femurs were taken for radiological, biomechanical, histological, and immunohistochemical analysis. Further, whole blood of the rat was incubated with granules and expression of 29 peptide mediators was assessed. Smallest granules showed significantly improved bone healing compared to larger granules, which however did not lead to an increased biomechanical stability in the defect zone. Small granules lead to an increased accumulation of macrophages in situ which could be assigned to the inflammatory subtype M1 by majority. Increased release of chemotactic respectively proangiogenic active factors in vitro compared to syngenic bone and beta-TCP was observed. Granule size of the bone graft substitute Herafill® has significant impact on bone healing of a critical size defect in combination with Masquelet's technique in terms of bone formation and inflammatory potential., (© 2019 The Authors. Journal of Biomedical Materials Research Part B: Applied Biomaterials published by Wiley Periodicals, Inc.)

Introduction: Cell-based therapy by bone marrow mononuclear cells (BMC) in a large-sized bone defect has already shown improved vascularization and new bone formation. First clinical trials are already being conducted. BMC were isolated from bone marrow aspirate and given back to patients in combination with a scaffold within some hours. However, the optimal concentration of BMC has not yet been determined for bone healing. With this study, we want to determine the optimal dosage of the BMC in the bone defect to support bone healing., Material and Methods: Scaffolds with increasing BMC concentrations were inserted into a 5 mm femoral defect, cell concentrations of 2 × 10 6 BMC/mL, 1 × 10 7 BMC/mL and 2 × 10 7 BMC/mL were used. Based on the initial cell number used to colonize the scaffolds, the groups are designated 1 × 10 6 , 5 × 10 6 and 1 × 10 7 group. Bone healing was assessed biomechanically, radiologically (µCT), and histologically after 8 weeks healing time., Results: Improved bone healing parameters were noted in the 1 × 10 6 and 5 × 10 6 BMC groups. A significantly higher BMD was observed in the 1 × 10 6 BMC group compared to the other groups. Histologically, a significantly increased bone growth in the defect area was observed in group 5 × 10 6 BMC. This finding could be supported radiologically., Conclusion: It was shown that the effective dose of BMC for bone defect healing ranges from 2 × 10 6 BMC/mL to 1 × 10 7 BMC/mL. This concentration range seems to be the therapeutic window for BMC-supported therapy of large bone defects. However, further studies are necessary to clarify the exact BMC-dose dependent mechanisms of bone defect healing and to determine the therapeutically effective range more precisely.

Introduction: The induced membrane technique for the treatment of large bone defects is a two-step procedure. In the first operation, a foreign body membrane is induced around a spacer, then, in the second step, several weeks or months later, the spacer is removed and the Membrane pocket is filled with autologous bone material. Induction of a functional biological membrane might be avoided by initially using a biological membrane. In this study, the effect of a human acellular dermis (hADM, Epiflex, DIZG gGmbH) was evaluated for the treatment of a large (5 mm), plate-stabilised femoral bone defect., Material and Methods: In an established rat model, hADM was compared to the two-stage induced membrane technique and a bone defect without membrane cover. Syngeneous spongiosa from donor animals was used for defect filling in all groups. The group size in each case was n = 5, the induction time of the membrane was 3-4 weeks and the healing time after filling of the defect was 8 weeks., Results: The ultimate loads were increased to levels comparable with native bone in both membrane groups (hADM: 63.2% ± 29.6% of the reference bone, p < 0.05 vs. no membrane, induced membrane: 52.1% ± 25.8% of the reference bone, p < 0.05 vs. no membrane) and were significantly higher than the control group without membrane (21.5%). The membrane groups were radiologically and histologically almost completely bridged by new bone formation, in contrast to the control Group where no closed osseous bridging could be observed., Conclusion: The use of the human acellular dermis leads to equivalent healing results in comparison to the two-stage induced membrane technique. This could lead to a shortened therapy duration of large bone defects.

Purpose: Treatment of complex fractures in the elderly is a challenge for operative reconstruction due to degraded bone structure. Early peri-operative bone anabolic treatment could improve new bone formation, avoid implant loosening and accelerate fracture healing., Methods: To compare the osteoanabolic potential of different drugs after distraction osteogenesis, 168 female Sprague-Dawley rats underwent lengthening of the right femur using a monolateral external fixator. Animals were randomly divided into six groups: vehicle-injected group, PTH(1-34), raloxifen, strontium ranelate, alendronate and simvastatin. Histomorphometry, CT-scanning, DEXA- and biomechanical analysis were performed to evaluate new bone formation, callus volume, mineralisation and biomechanical strength. Expression of bone metabolic mediators and differentiation indicators of distracted and intact bone were examined by RT-PCR and western blot., Results: Histological analysis showed significant increase of the bone mass after treatment with PTH(1-34), raloxifen and strontium ranelate (p = 0.02). Raloxifen increased bone mineral content (BMC) of the whole distracted femur significantly (p = 0.007). Callus volume was significantly larger in the PTH(1-34), raloxifen and simvastatin groups (p = 0.001) compared to control. Ultimate load of distracted new formed bone was increased in PTH(1-34) and raloxifen groups. It seems that PTH(1-34) and raloxifen have a stronger effect on bone where a repair response is activated. Strontium ranelate demonstrates similar effects to PTH regarding new bone formation but shows low values for mineralisation and biomechanical strength., Conclusion: This study suggests that peri-operative treatment of complex and/or osteoporotic fractures with PTH(1-34) and raloxifen might be useful as a stimulator of bone formation and mineralisation to shorten the consolidation time in humans.

Large segmental bone defects occurring after trauma, bone tumors, infections or revision surgeries are a challenge for surgeons. The aim of our study was to develop a new biomaterial utilizing simple and cheap 3D-printing techniques. A porous polylactide (PLA) cylinder was printed and functionalized with stromal-derived factor 1 (SDF-1) or bone morphogenetic protein 7 (BMP-7) immobilized in collagen type I. Biomechanical testing proved biomechanical stability and the scaffolds were implanted into a 6 mm critical size defect in rat femur. Bone growth was observed via x-ray and after 8 weeks, bone regeneration was analyzed with µCT and histological staining methods. Development of non-unions was detected in the control group with no implant. Implantation of PLA cylinder alone resulted in a slight but not significant osteoconductive effect, which was more pronounced in the group where the PLA cylinder was loaded with collagen type I. Addition of SDF-1 resulted in an osteoinductive effect, with stronger new bone formation. BMP-7 treatment showed the most distinct effect on bone regeneration. However, histological analyses revealed that newly formed bone in the BMP-7 group displayed a holey structure. Our results confirm the osteoinductive character of this 3D-biofabricated cell-free new biomaterial and raise new options for its application in bone tissue regeneration.

Large bone defects often pose major difficulties in orthopaedic surgery. The application of long-term cultured stem cells combined with a scaffold lead to a significant improvement of bone healing in recent experiments but is strongly restricted by European Union law. Bone marrow mononuclear cells (BMC), however, can be isolated and transplanted within a few hours and have been proven effective in experimental models of bone healing. The effectivity of the BMC-supported therapy might be influenced by the type of scaffold. Hence, we compared three different scaffolds serving as a carrier for BMC in a rat femoral critical size defect with regard to the osteogenic activity in the defect zone. Human demineralized bone matrix (DBM), bovine cancellous bone hydroxyapatite ceramic (BS), or β-tricalcium phosphate (β-TCP) were seeded with human BMC and hereafter implanted into critically sized bone defects of male athymic nude rats. Autologous bone served as a control. Gene activity was measured after 1 week, and bone formation was analysed histologically and radiologically after 8 weeks. Generally, regenerative gene expression (BMP2, RUNX2, VEGF, SDF-1, and RANKL) as well as bony bridging and callus formation was observed to be most pronounced in defects filled with autologous bone, followed in descending order by DBM, β-TCP, and BS. Although DBM was superior in most aspects of bone regeneration analysed in comparison to β-TCP and BS, the level of autologous bone could not be attained., (Copyright © 2017 John Wiley & Sons, Ltd.)

Vascularized periosteal flaps are used for complex cases if the reconstruction of large bone defects is necessary in modern trauma and orthopedic surgery. In this study, we combined this surgical procedure with β‑TCP scaffold and mesenchymal stem cells (MSCs) + endothelial progenitor cells (EPCs) as a tissue engineering approach to obtain optimum conditions for bone healing in rats. A critical size femoral defect was created in 80 rats allocated into 4 groups. Defects were treated according to the following protocol: i) vascularized periosteal flap alone; ⅱ) vascularized periosteal flap + β‑TCP scaffold; ⅲ) vascularized periosteal flap + β‑TCP scaffold + ligated vascular pedicle; and ⅳ) vascularized periosteal flap + β‑TCP scaffold + MSCs/EPCs. After 8 weeks, femur bones were extracted and analyzed for new bone formation, vascularization, proliferation and inflammatory processes and strength. Bone mineral density (BMD) and biomechanical stability at week 8 were highest in group 4 (flap + β‑TCP scaffold + MSCs/EPCs) compared to all the other groups. Stability was significantly higher in group 4 (flap + β‑TCP scaffold + MSCs/EPCs) in comparison to group 3 (ligated flap + β‑TCP scaffold). BMD was found to be significantly lower in group 3 (ligated flap + β‑TCP scaffold) compared to group 1 (flap) and group 4 (flap + β‑TCP scaffold + MSCs/EPCs). The highest density of blood vessels was observed in group 4 (flap + β‑TCP + MSCs/EPCs) and the values were significantly increased in comparison to group 3 (ligated flap), but not to group 1 (flap) and group 2 (flap + β‑TCP). The highest amounts of proliferating cells were observed in group 4 (flap + β‑TCP scaffold + MSC/EPCs). The percentage of proliferating cells was significantly higher in group 4 (flap + β‑TCP scaffold + MSCs/EPCs) in comparison to all the other groups after 8 weeks. Our data thus indicate that critical size defect healing could be improved if MSCs/EPCs are added to β-TCP scaffold in combination with a periosteal flap. Even after 8 weeks, the amount of proliferating cells was increased. The flap blood supply is essential for bone healing and the reduction of inflammatory processes.

Introduction: The surgical treatment of large bone defects continues to pose a major challenge in modern trauma and orthopedic surgery. In this study we test the effectiveness of a tissue engineering approach, using three-dimensional (3D) β-tricalcium phosphate (β-TCP) scaffolding plus bone marrow-derived mononuclear cells (BM-MNCs), combined with a vascularized periosteal flap, in a rat femur critical size defect model., Methods: Eighty rats were randomly allocated into four equal groups. Under general anesthesia, critical size defects were created on their femurs and were treated with (1) Vascularized periosteal flap alone, (2) Vascularized periosteal flap+β-TCP scaffolding, (3) Vascularized periosteal flap+β-TCP scaffolding+ligated vascular pedicle, and (4) Vascularized periosteal flap+β-TCP scaffolding+BM-MNCs. After 4 and 8 weeks animals were euthanized and the bone defects were harvested for analysis of new bone formation, vascularization, and strength using histology, immunohistology, micro-CT, and biomechanical testing, respectively., Results: Group 1: (P. flap) Increase in new bone formation and vascularization. Group 2: (P. flap+scaffold) Increase in new bone formation and vascularization. Group 3: (P. flap+scaffold+ligated vascular pedicle) No new bone formation and no vascularization. Group 4: (P. flap+scaffold+BM-MNCs) A significant (p < 0.05) increase was seen in new bone formation, vascularization, and strength in bones treated with flaps, scaffold, and BM-MNCs, when compared with the other treatment groups., Conclusion: Combining a vascularized periosteal flap with tissue engineering approach (β-TCP scaffolding and BM-MNC) results in significantly improved bone healing in our rat femur critical size bone defect model.

Unlabelled: QUESTION/AIM: Cell-based therapy by cultivated stem cells (mesenchymal stem cells [MSC] and endothelial progenitor cells [EPC]) in a large-sized bone defect has already shown improved vascularization and new bone formation. However, these methods are clinically afflicted with disadvantages. Another heterogeneous bone marrow cell population, the so-called human bone marrow-derived mononuclear cells (BMC), has nevertheless been used clinically and showed improved vascularization in ischemic limbs or in the myocardium. For clinical use, a certified process has been established; thus, BMC were isolated from bone marrow aspirate by density gradient centrifugation, washed, cleaned, and given back to patients within several hours. This investigation tested the ability of human BMC seeded on beta-tricalcium phosphate (β-TCP) and placed into a large bone defect in rats to improve the bone healing process in vivo., Methods: Human EPC were isolated from buffy coat, and MSC or BMC, respectively, were isolated from bone marrow aspirate by density gradient centrifugation. 1.0×10(6) cells were loaded onto 750 μL β-TCP (0.7-1.4 mm). Large femoral defects (6 mm) in athymic rats were created surgically and stabilized with an internal fixateur. The remaining defects were filled with β-TCP granules alone (group 1), β-TCP+EPC/MSC (group 2), or β-TCP+BMC (group 3). After 8 weeks, histomorphometric analysis (new bone formation), radiological microcomputer tomography analysis (bony bridging), and biomechanical testing (three-point bending) were achieved. Moreover, a tumorigenicity study was performed to evaluate the safety of BMC implantation after 26 weeks. For statistical analysis, the Kruskal-Wallis test was used., Results: Eight weeks after implantation of EPC/MSC or BMC, respectively, we detected a more significant new bone formation compared to control. In group 2 and 3, bony bridging of the defect was seen. In the control group, more chondrocytes and osteoid were detected. In the BMC and EPC/MSC group, respectively, less chondrocytes and a significantly more advanced bone formation were observed. The biomechanical stability of the bone regenerate was significantly enhanced if BMC and EPC/MSC, respectively, were implanted compared to control. Moreover, no tumor formation was detected either macroscopically or histologically after 26 weeks of BMC implantation., Discussion: Implanted BMC suggest that a heterogeneous cell population may provide a powerful cellular therapeutic strategy for bone healing in a large bone defect in humans.

Autologous bone grafting represents the gold standard modality to treat atrophic non-unions by virtue of its osteoinductive and osteoconductive properties. The common harvest site is the iliac crest, but there are major concerns due to limited volume and considerable donor site morbidity. Alternative autologous bone graft can be harvested from the femoral bone cavity using a newly developed 'Reamer Irrigator Aspirator' (RIA). Osseous aspirated particles can be recovered with a filter and used as auto-graft. The purpose of this study was to compare the concentration and differentiation potential of mesenchymal stem cells (MSC) and endothelial progenitor cells (EPC) harvested with the RIA technique or from the iliac crest, respectively. RIA aspirate was collected from 26 patients undergoing intramedullary nailing of femur fractures. Iliac crest aspirate was collected from 38 patients undergoing bone graft transplantation. Concentration of MSC and EPC were assessed by means of the MSC colony assay, EPC culture assay and flowcytometry (CD34, CD133, VEGF-R2), respectively. Osteogenic differentiation of MSC's was measured by von Kossa staining. Patients in both groups did not significantly differ regarding their age, gender or pre-existing health conditions. In comparison to aspirates obtained from iliac crest the RIA aspirates from the femur contained a significantly higher percentage of CD34+ progenitor cells, a significantly higher concentration of MSC and a significantly higher concentration of early EPC. The percentage of late EPC did not differ between both sites. Moreover, the capability of MSC for calcium deposition was significantly enhanced in MSC obtained with RIA. Our results show that RIA aspirate is a rich source for different types of autologous progenitor cells, which can be used to accelerate healing of bone and other musculoskeletal tissues., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Critical-size bone defects up to 25 cm can be treated successfully using the induced membrane technique established by Masquelet. To shorten this procedure, human acellular dermis (HAD) has had success in replacing this membrane in rat models. The aim of this study was to compare bone healing for smaller and larger defects using an induced membrane and HAD in a rat model. Using our established femoral defect model in rats, the animals were placed into four groups and defects of 5 mm or 10 mm size were set, either filling them with autologous spongiosa and surrounding the defect with HAD or waiting for the induced membrane to form around a cement spacer and filling this cavity in a second operation with a cancellous bone graft. Healing was assessed eight weeks after the operation using µ-CT, histological staining, and an assessment of the progress of bone formation using an established bone healing score. The α-smooth muscle actin used as a signal of blood vessel formation was stained and counted. The 5 mm defects showed significantly better bone union and a higher bone healing score than the 10 mm defects. HAD being used for the smaller defects resulted in a significantly higher bone healing score even than for the induced membrane and significantly higher blood vessel formation, corroborating the good results achieved by using HAD in previous studies. In comparison, same-sized groups showed significant differences in bone healing as well as blood vessel formation, suggesting that 5 mm defects are large enough to show different results in healing depending on treatment; therefore, 5 mm is a viable size for further studies on bone healing. [ABSTRACT FROM AUTHOR]

The classic two-stage masquelet technique is an effective procedure for the treatment of large bone defects. Our group recently showed that one surgery could be saved by using a decellularized dermis membrane (DCD, Epiflex, DIZG). In addition, studies with bone substitute materials for defect filling show that it also appears possible to dispense with the removal of syngeneic cancellous bone (SCB), which is fraught with complications. The focus of this work was to clarify whether the SCB can be replaced by the granular demineralized bone matrix (g-DBM) or fibrous demineralized bone matrix (f-DBM) demineralized bone matrix and whether the colonization of the DCD and/or the DBM defect filling with bone marrow mononuclear cells (BMC) can lead to improved bone healing. In 100 Sprague Dawley rats, a critical femoral bone defect 5 mm in length was stabilized with a plate and then encased in DCD. Subsequently, the defect was filled with SCB (control), g-DBM, or f-DBM, with or without BMC. After 8 weeks, the femurs were harvested and subjected to histological, radiological, and biomechanical analysis. The analyses showed the incipient bony bridging of the defect zone in both groups for g-DBM and f-DBM. Stability and bone formation were not affected compared to the control group. The addition of BMCs showed no further improvement in bone healing. In conclusion, DBM offers a new perspective on defect filling; however, the addition of BMC did not lead to better results. [ABSTRACT FROM AUTHOR]

The Masquelet technique is used to treat large bone defects; it is a two-stage procedure based on an induced membrane. To improve the induced membrane process, demineralized bone matrix in granular (GDBM) and fibrous form (f-DBM) was tested with and without bone marrow mononuclear cells (BMC) as filling of the membrane against the gold standard filling with syngeneic cancellous bone (SCB). A total of 65 male Sprague–Dawley rats obtained a 5 mm femoral defect. These defects were treated with the induced membrane technique and filled with SCB, GDBM, or f-DBM, with or without BMC. After a healing period of eight weeks, the femurs were harvested and submitted for histological, radiological, and biomechanical analyses. The fracture load in the defect zone was lower compared to SCB in all groups. However, histological analysis showed comparable new bone formation, bone mineral density, and cartilage proportions and vascularization. The results suggest that f-DBM in combination with BMC and the induced membrane technique cannot reproduce the very good results of this material in large, non-membrane coated bone defects, nevertheless it supports the maturation of new bone tissue locally. It can be concluded that BMC should be applied in lower doses and inflammatory cells should be removed from the cell preparation before implantation. [ABSTRACT FROM AUTHOR]

Regeneration of large bone defects is a major objective in trauma surgery. Bone marrow mononuclear cell (BMC)-supported bone healing was shown to be efficient after immobilization on a scaffold. We hypothesized that fibrous demineralized bone matrix (DBM) in various forms with BMCs is superior to granular DBM. A total of 65 male SD rats were assigned to five treatment groups: syngenic cancellous bone (SCB), fibrous demineralized bone matrix (f-DBM), fibrous demineralized bone matrix densely packed (f-DBM 120%), DBM granules (GDBM) and DBM granules 5% calcium phosphate (GDBM5%Ca2+). BMCs from donor rats were combined with different scaffolds and placed into 5 mm femoral bone defects. After 8 weeks, bone mineral density (BMD), biomechanical stability and histology were assessed. Similar biomechanical properties of f-DBM and SCB defects were observed. Similar bone and cartilage formation was found in all groups, but a significantly bigger residual defect size was found in GDBM. High bone healing scores were found in f-DBM (25) and SCB (25). The application of DBM in fiber form combined with the application of BMCs shows promising results comparable to the gold standard, syngenic cancellous bone. Denser packing of fibers or higher amount of calcium phosphate has no positive effect. [ABSTRACT FROM AUTHOR]