Arne Berner

A Tissue Engineering Solution for Segmental Defect Regeneration in Load-Bearing Long Bones

A polycaprolactone-tricalcium phosphate scaffold with recombinant human BMP-7 heals critical-sized bone defects in sheep. Building Up Bone Large gaps or defects in bone are typically bridged using segments of bone from elsewhere in the body [referred to as autologous bone grafts (ABGs)]. It is not ideal, however, to harvest bone tissue from elsewhere; it is two surgeries, two defect sites, and therefore an increased risk of infection. Instead, tissue engineers have taken on this challenge of replenishing lost bone. In this issue, Reichert and colleagues have designed a polymer-based scaffold that can be loaded with cells and growth factors and inserted directly into a bone defect, with healing demonstrated in sheep after only 3 months. Reichert et al. used their medical-grade polycaprolactone–tricalcium phosphate (mPCL-TCP) scaffolds either alone or in combination with donor mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). The scaffolds were implanted into critical-sized defects (3 cm) in the long bones of sheep, whose bones resemble formation and structure in humans, and are therefore a good model for bone tissue regeneration. After 3 months, the authors reported bone bridging in 100% of the ABGs and scaffold/rhBMP-7 groups but saw bridging in only 38% of the bare scaffold and scaffold/MSC groups. After 12 months, however, animals treated with the scaffold/rhBMP-7 combination showed greater bone volume and mechanical strength than the ABG positive control. The authors attribute this improvement over time to be the result of local BMP delivery (greater stimulation of bone formation) in addition to more bone deposition along the periphery of the defect (enhanced strength). The addition of MSCs did not help bone regeneration, as other studies have shown previously. The next step is determining the ideal BMP dose and the mechanism underlying the effects of the scaffold/rhBMP-7 on surrounding cells and tissue. Then, the hope is to move to clinical trials, where this scaffold will be put to the test for evaluation of bone regeneration and load bearing in humans. The reconstruction of large defects (>10 mm) in humans usually relies on bone graft transplantation. Limiting factors include availability of graft material, comorbidity, and insufficient integration into the damaged bone. We compare the gold standard autograft with biodegradable composite scaffolds consisting of medical-grade polycaprolactone and tricalcium phosphate combined with autologous bone marrow–derived mesenchymal stem cells (MSCs) or recombinant human bone morphogenetic protein 7 (rhBMP-7). Critical-sized defects in sheep—a model closely resembling human bone formation and structure—were treated with autograft, rhBMP-7, or MSCs. Bridging was observed within 3 months for both the autograft and the rhBMP-7 treatment. After 12 months, biomechanical analysis and microcomputed tomography imaging showed significantly greater bone formation and superior strength for the biomaterial scaffolds loaded with rhBMP-7 compared to the autograft. Axial bone distribution was greater at the interfaces. With rhBMP-7, at 3 months, the radial bone distribution within the scaffolds was homogeneous. At 12 months, however, significantly more bone was found in the scaffold architecture, indicating bone remodeling. Scaffolds alone or with MSC inclusion did not induce levels of bone formation comparable to those of the autograft and rhBMP-7 groups. Applied clinically, this approach using rhBMP-7 could overcome autograft-associated limitations.

Role of mesenchymal stem cells in tissue engineering of meniscus

Tissue engineering is a promising approach for the treatment of tissue defects. Mesenchymal stem cells are of potential use as a source of repair cells or of important growth factors for tissue engineering. The purpose of this study was to examine the role of mesenchymal stem cells in meniscal tissue repair. This was tested using several cell and biomaterial-based treatment options for repair of defects in the avascular zone of rabbit menisci. Circular meniscal punch defects (2 mm) were created in the avascular zone of rabbit menisci and left empty or filled with hyaluronan-collagen composite matrices without cells, loaded with platelet-rich plasma, autologous bone marrow, or autologous mesenchymal stem cells. In some experiments, matrices with stem cells were precultured in chondrogenic medium for 14 days before implantation. Rabbits were then allowed free cage movement after surgery for up to 12 weeks. Untreated defects and defects treated with cell-free implants had muted fibrous healing responses. Neither bone marrow nor platelet-rich plasma loaded in matrices produced improvement in healing compared with cell-free implants. The implantation of 14 days precultured chondrogenic stem cell-matrix constructs resulted in fibrocartilage-like repair tissue, which was only partially integrated with the native meniscus. Non-precultured mesenchymal stem cells in hyaluronan-collagen composite matrices stimulated the development of completely integrated meniscus-like repair tissue. The study shows the necessity of mesenchymal stem cells for the repair of meniscal defects in the avascular zone. Mesenchymal stem cells seem to fulfill additional repair qualities besides the delivery of growth factors.

Bone tissue engineering: from bench to bedside

The drive to develop bone grafts for the filling of major gaps in the skeletal structure has led to a major research thrust towards developing biomaterials for bone engineering. Unfortunately, from a clinical perspective, the promise of bone tissue engineering which was so vibrant a decade ago has so far failed to deliver the anticipated results of becoming a routine therapeutic application in reconstructive surgery. Here we describe our bench to bedside concept, the first clinical results and a detailed analysis of long-term bone regeneration studies in preclinical animal models, exploiting methods of micro- and nano analysis of biodegradable composite scaffolds.

Autologous vs. allogenic mesenchymal progenitor cells for the reconstruction of critical sized segmental tibial bone defects in aged sheep

Mesenchymal progenitor cells (MPCs) represent an attractive cell population for bone tissue engineering. Their special immunological characteristics suggest that MPCs may be used in allogenic applications. The objective of this study was to compare the regenerative potential of autologous vs. allogenic MPCs in an ovine critical size segmental defect model. Ovine MPCs were isolated from bone marrow aspirates, expanded and cultured with osteogenic medium for 2weeks before implantation. Autologous and allogenic transplantation was performed using the cell-seeded scaffolds and unloaded scaffolds, while the application of autologous bone grafts served as a control group (n=6). Bone healing was assessed 12weeks after surgery by radiology, microcomputed tomography, biomechanical testing and histology. Radiology, biomechanical testing and histology revealed no significant differences in bone formation between the autologous and allogenic groups. Both cell groups showed more bone formation than the scaffold alone, whereas the biomechanical data showed no significant differences between the cell groups and the unloaded scaffolds. The results of the study suggest that scaffold-based bone tissue engineering using allogenic cells offers the potential for an off-the-shelf product. Thus the results of this study serve as an important baseline for translation of the assessed concepts into clinical applications.

Angiogenesis: The role of PDGF-BB on Adiopse-tissue derived Stem Cells (ASCs)

Recently, it was shown that mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells which highlights the potential role of MSCs in neovascularization. In the present study, we investigated the paracrine factors responsible for tube formation in human adipose-tissue derived stem cells (ASCs). Moreover, we analyzed ASCs migration towards PDGF-BB and altered levels of proteins involved in different pathways. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta (PDGF) or basic fibroblast growth factor (bFGF). Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 139 phosphorylated or native proteins after incubation with PDGF-BB protein for 24 hours. The present data suggest, that freshly isolated ASCs contain a subpopuplation of stem cells that can form capillary like tubes which is dependent on PDGF and bFGF signaling pathway. Furthermore, Migration of human ASCs significantly increased in response to increased concentrations of PDGF-BB. In addition, incubation of ASCs with PDGF-BB altered phosphorylation of several transcription proteins that are widely expressed throughout the hematopoietic system, targeting genes that have been associated with proliferation, anti-apoptosis or differentiation.

Treatment of long bone defects and non-unions: from research to clinical practice

The treatment of long bone defects and non-unions is still a major clinical and socio-economical problem. In addition to the non-operative therapeutic options, such as the application of various forms of electricity, extracorporeal shock wave therapy and ultrasound therapy, which are still in clinical use, several operative treatment methods are available. No consensus guidelines are available and the treatments of such defects differ greatly. Therefore, clinicians and researchers are presently investigating ways to treat large bone defects based on tissue engineering approaches. Tissue engineering strategies for bone regeneration seem to be a promising option in regenerative medicine. Several in vitro and in vivo studies in small and large animal models have been conducted to establish the efficiency of various tissue engineering approaches. Neverthelsss, the literature still lacks controlled studies that compare the different clinical treatment strategies currently in use. However, based on the results obtained so far in diverse animal studies, bone tissue engineering approaches need further validation in more clinically relevant animal models and in clinical pilot studies for the translation of bone tissue engineering approaches into clinical practice.

Fabrication of polycaprolactone collagen hydrogel constructs seeded with mesenchymal stem cells for bone regeneration

The osteogenic differentiation of bone marrow-derived human mesenchymal stem cells (MSCs) in a collagen I hydrogel was investigated. Collagen hydrogels with 7.5 x 10(5) MSCs ml(-1) were fabricated and cultured for 6 weeks in a defined, osteogenic differentiation medium. Histochemistry revealed morphologically distinct, chondrocyte-like cells, surrounded by a sulfated proteoglycan-rich extracellular matrix in the group treated with bone morphogenetic protein 2 (BMP-2), while cells cultured with dexamethasone, ascorbate-2-phosphate, and beta-glycerophosphate displayed a spindle-shaped morphology and deposited a mineralized matrix. Real-time polymerase chain reaction (RT-PCR) analyses revealed a specific chondrogenic differentiation with the expression of cartilage-specific markers in the BMP-2-treated group and a distinct expression pattern of the osteogenic markers alkaline phosphatase (ALP), type I collagen, osteocalcin (OC), and cbfa-1 in the group treated with an osteogenic standard medium. The collagen gels were used to engineer a cell laden medical grade epsilon-polycaprolactone (PCL)-hydrogel construct for segmental bone repair showing good bonding at the scaffold hydrogel interface and even cell distribution. The results show that MSCs cultured in a collagen I hydrogel are able to undergo a distinct osteogenic differentiation pathway when stimulated with specific differentiation factors and suggest that collagen I hydrogels are a suitable means to facilitate cell seeding of scaffolds for bone tissue engineering applications.

Are Applied Growth Factors Able to Mimic the Positive Effects of Mesenchymal Stem Cells on the Regeneration of Meniscus in the Avascular Zone

Meniscal lesions in the avascular zone are still a problem in traumatology. Tissue Engineering approaches with mesenchymal stem cells (MSCs) showed successful regeneration of meniscal defects in the avascular zone. However, in daily clinical practice, a single stage regenerative treatment would be preferable for meniscus injuries. In particular, clinically applicable bioactive substances or isolated growth factors like platelet-rich plasma (PRP) or bone morphogenic protein 7 (BMP7) are in the focus of interest. In this study, the effects of PRP and BMP7 on the regeneration of avascular meniscal defects were evaluated. In vitro analysis showed that PRP secretes multiple growth factors over a period of 8 days. BMP7 enhances the collagen II deposition in an aggregate culture model of MSCs. However applied to meniscal defects PRP or BMP7 in combination with a hyaluronan collagen composite matrix failed to significantly improve meniscus healing in the avascular zone in a rabbit model after 3 months. Further information of the repair mechanism at the defect site is needed to develop special release systems or carriers for the appropriate application of growth factors to support biological augmentation of meniscus regeneration.

Screw placement in percutaneous acetabular surgery: gender differences of anatomical landmarks in a cadaveric study.

PurposePercutaneous reduction and periarticular screw implantation techniques have been successfully introduced in acetabular surgery. The advantages of this less invasive approach are attenuated by higher risks of screw misplacement. Anatomical landmarks are strongly needed to prevent malplacement. This cadaver study was designed to identify reliable anatomical osseous landmarks in the pelvic region for screw placement in acetabular surgery. Gender differences were specifically addressed.MethodsTwenty-seven embalmed cadaveric hemipelvic specimens (13 male, 14 female) were used. After soft-tissue removal, anterior and posterior column acetabular screw placement was conducted by one orthopaedic trauma surgeon under direct vision. Each column was addressed by antegrade and retrograde screw insertion. Radiographic verification of ideal screw placement was followed by assessment of the distance from the different entry points to adjoining anatomical osseous structures.ResultsFor anterior column screw positioning, the posterior superior iliac spine (PSIS), posterior inferior iliac spine (PIIS), iliopectineal eminence and centre of the symphysis were most reliable regarding gender differences. For posterior column screw positioning, the distance to the anterior superior iliac spine (ASIS) and the ischial tuberosity showed the lowest deviation between the different gender specimens. Highest gender differences were seen in relation to the cranial rim of the superior pubic ramus in retrograde anterior column screw positioning (p = 0.002). Most landmarks could be targeted within a 2.5-cm range in all specimens.ConclusionsThe findings emphasise the relevance of osseous landmarks in acetabular surgery. By adhering to easily identifiable structures, screw placement can be safely performed. Significant gender differences must be taken into consideration during preoperative planning.

Poly(ε-caprolactone) Scaffolds Fabricated by Melt Electrospinning for Bone Tissue Engineering

Melt electrospinning is a promising approach to manufacture biocompatible scaffolds for tissue engineering. In this study, melt electrospinning of poly(ε-caprolactone) onto structured, metallic collectors resulted in scaffolds with an average pore size of 250–300 μm and an average fibre diameter of 15 μm. Scaffolds were seeded with ovine osteoblasts in vitro. Cell proliferation and deposition of mineralised extracellular matrix was assessed using PicoGreen® (Thermo Fisher Scientific, Scoresby, Australia) and WAKO® HR II (WAKO, Osaka, Japan) calcium assays. Biocompatibility, cell infiltration and the growth pattern of osteoblasts on scaffolds was investigated using confocal microscopy and scanning electron microscopy. Osteoblasts proliferated on the scaffolds over an entire 40-day culture period, with excellent survival rates and deposited mineralized extracellular matrix. In general, the 3D environment of the structured melt electrospun scaffold was favourable for osteoblast cultures.