Johannes C. Reichert

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.

Translating tissue engineering technology platforms into cancer research

•  Introduction •  History of tissue engineering •  Physiological and structural aspects of 2D versus 3D culture in cancer research •  State of the art of 3D culture systems in cancer research •  New tissue engineering‐routed scaffolds for 3D culture •  Endothelial progenitor cells and tumour vasculature •  In vivo models •  Arteriovenous loop isolation chamber for tumour angiogenesis research •  Conclusion

Strategies for Zonal Cartilage Repair using Hydrogels

Articular cartilage is a highly hydrated tissue with depth-dependent cellular and matrix properties that provide low-friction load bearing in joints. However, the structure and function are frequently lost and there is insufficient repair response to regenerate high-quality cartilage. Several hydrogel-based tissue-engineering strategies have recently been developed to form constructs with biomimetic zonal variations to improve cartilage repair. Modular hydrogel systems allow for systematic control over hydrogel properties, and advanced fabrication techniques allow for control over construct organization. These technologies have great potential to address many unanswered questions involved in prescribing zonal properties to tissue-engineered constructs for cartilage repair.

Mineralized human primary osteoblast matrices as a model system to analyse interactions of prostate cancer cells with the bone microenvironment

Prostate cancer metastasis is reliant on the reciprocal interactions between cancer cells and the bone niche/micro-environment. The production of suitable matrices to study metastasis, carcinogenesis and in particular prostate cancer/bone micro-environment interaction has been limited to specific protein matrices or matrix secreted by immortalised cell lines that may have undergone transformation processes altering signaling pathways and modifying gene or receptor expression. We hypothesize that matrices produced by primary human osteoblasts are a suitable means to develop an in vitro model system for bone metastasis research mimicking in vivo conditions. We have used a decellularized matrix secreted from primary human osteoblasts as a model for prostate cancer function in the bone micro-environment. We show that this collagen I rich matrix is of fibrillar appearance, highly mineralized, and contains proteins, such as osteocalcin, osteonectin and osteopontin, and growth factors characteristic of bone extracellular matrix (ECM). LNCaP and PC3 cells grown on this matrix, adhere strongly, proliferate, and express markers consistent with a loss of epithelial phenotype. Moreover, growth of these cells on the matrix is accompanied by the induction of genes associated with attachment, migration, increased invasive potential, Ca(2+) signaling and osteolysis. In summary, we show that growth of prostate cancer cells on matrices produced by primary human osteoblasts mimics key features of prostate cancer bone metastases and thus is a suitable model system to study the tumor/bone micro-environment interaction in this disease.

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.

Porous scaffold architecture guides tissue formation

Critical‐sized bone defect regeneration is a remaining clinical concern. Numerous scaffold‐based strategies are currently being investigated to enable in vivo bone defect healing. However, a deeper understanding of how a scaffold influences the tissue formation process and how this compares to endogenous bone formation or to regular fracture healing is missing. It is hypothesized that the porous scaffold architecture can serve as a guiding substrate to enable the formation of a structured fibrous network as a prerequirement for later bone formation. An ovine, tibial, 30‐mm critical‐sized defect is used as a model system to better understand the effect of the scaffold architecture on cell organization, fibrous tissue, and mineralized tissue formation mechanisms in vivo. Tissue regeneration patterns within two geometrically distinct macroscopic regions of a specific scaffold design, the scaffold wall and the endosteal cavity, are compared with tissue formation in an empty defect (negative control) and with cortical bone (positive control). Histology, backscattered electron imaging, scanning small‐angle X‐ray scattering, and nanoindentation are used to assess the morphology of fibrous and mineralized tissue, to measure the average mineral particle thickness and the degree of alignment, and to map the local elastic indentation modulus. The scaffold proves to function as a guiding substrate to the tissue formation process. It enables the arrangement of a structured fibrous tissue across the entire defect, which acts as a secondary supporting network for cells. Mineralization can then initiate along the fibrous network, resulting in bone ingrowth into a critical‐sized defect, although not in complete bridging of the defect. The fibrous network morphology, which in turn is guided by the scaffold architecture, influences the microstructure of the newly formed bone. These results allow a deeper understanding of the mode of mineral tissue formation and the way this is influenced by the scaffold architecture.

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.

Long-term effects of hydrogel properties on human chondrocyte behavior

Hydrogels provide a 3-dimensional network for embedded cells and offer promise for cartilage tissue engineering applications. Nature-derived hydrogels, including alginate, have been shown to enhance the chondrocyte phenotype but are variable and not entirely controllable. Synthetic hydrogels, including polyethylene glycol (PEG)-based matrices, have the advantage of repeatability and modularity; mechanical stiffness, cell adhesion, and degradability can be altered independently. In this study, we compared the long-term in vitro effects of different hydrogels (alginate and Factor XIIIa-cross-linked MMP-sensitive PEG at two stiffness levels) on the behavior of expanded human chondrocytes and the development of construct properties. Monolayer-expanded human chondrocytes remained viable throughout culture, but morphology varied greatly in different hydrogels. Chondrocytes were characteristically round in alginate but mostly spread in PEG gels at both concentrations. Chondrogenic gene (COL2A1, aggrecan) expression increased in all hydrogels, but alginate constructs had much higher expression levels of these genes (up to 90-fold for COL2A1), as well as proteoglycan 4, a functional marker of the superficial zone. Also, chondrocytes expressed COL1A1 and COL10A1, indicative of de-differentiation and hypertrophy. After 12 weeks, constructs with lower polymer content were stiffer than similar constructs with higher polymer content, with the highest compressive modulus measured in 2.5% PEG gels. Different materials and polymer concentrations have markedly different potency to affect chondrocyte behavior. While synthetic hydrogels offer many advantages over natural materials such as alginate, they must be further optimized to elicit desired chondrocyte responses for use as cartilage models and for development of functional tissue-engineered articular cartilage.

Stem cell- and growth factor-based regenerative therapies for avascular necrosis of the femoral head

Avascular necrosis (AVN) of the femoral head is a debilitating disease of multifactorial genesis, predominately affects young patients, and often leads to the development of secondary osteoarthritis. The evolving field of regenerative medicine offers promising treatment strategies using cells, biomaterial scaffolds, and bioactive factors, which might improve clinical outcome. Early stages of AVN with preserved structural integrity of the subchondral plate are accessible to retrograde surgical procedures, such as core decompression to reduce the intraosseous pressure and to induce bone remodeling. The additive application of concentrated bone marrow aspirates, ex vivo expanded mesenchymal stem cells, and osteogenic or angiogenic growth factors (or both) holds great potential to improve bone regeneration. In contrast, advanced stages of AVN with collapsed subchondral bone require an osteochondral reconstruction to preserve the physiological joint function. Analogously to strategies for osteochondral reconstruction in the knee, anterograde surgical techniques, such as osteochondral transplantation (mosaicplasty), matrix-based autologous chondrocyte implantation, or the use of acellular scaffolds alone, might preserve joint function and reduce the need for hip replacement. This review summarizes recent experimental accomplishments and initial clinical findings in the field of regenerative medicine which apply cells, growth factors, and matrices to address the clinical problem of AVN.

Ovine bone- and marrow-derived progenitor cells and their potential for scaffold-based bone tissue engineering applications in vitro and in vivo.

Recently, research has focused on bone marrow derived multipotent mesenchymal precursor cells (MPC) and osteoblasts (OB) for clinical use in bone engineering. Prior to clinical application, cell based treatment concepts need to be evaluated in preclinical, large animal models. Sheep in particular are considered a valid model for orthopaedic and trauma related research. However, only sheep aged > 6 years show secondary osteon formation characteristic of human bone. Osteogenic cells isolated from animals of this age group remain poorly characterized. In the present study, ex vivo expanded MPC isolated from ovine bone marrow proliferated at a higher rate than OB derived from tibial compact bone as assessed in standard 2D cultures. MPC expressed the respective phenotypic profile typical for different mesenchymal cell populations (CD14−/CD31−/CD45−/CD29+/CD44+/CD166+) and showed a multilineage differentiation potential. When compared to OB, MPC had a higher mineralization potential under standard osteogenic culture conditions and expressed typical bone related markers such as osteocalcin, osteonectin and type I collagen at the mRNA and protein level. After 4 weeks in 3D culture, MPC constructs demonstrated higher cell density and mineralization, whilst cell viability on the scaffolds was assessed > 90%. Cells displayed a spindle‐like morphology and formed interconnected networks. In contrast, when implanted subcutaneously into NOD/SCID mice, MPC presented a lower osteogenic potential than OB. In summary, this study provides a detailed characterisation of ovine MPC and OB from a bone engineering perspective and suggests that MPC and OB provide promising means for future bone disease related treatment applications. Copyright