Manav Mehta

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.

Biomaterial delivery of morphogens to mimic the natural healing cascade in bone

Complications in treatment of large bone defects using bone grafting still remain. Our understanding of the endogenous bone regeneration cascade has inspired the exploration of a wide variety of growth factors (GFs) in an effort to mimic the natural signaling that controls bone healing. Biomaterial-based delivery of single exogenous GFs has shown therapeutic efficacy, and this likely relates to its ability to recruit and promote replication of cells involved in tissue development and the healing process. However, as the natural bone healing cascade involves the action of multiple factors, each acting in a specific spatiotemporal pattern, strategies aiming to mimic the critical aspects of this process will likely benefit from the usage of multiple therapeutic agents. This article reviews the current status of approaches to deliver single GFs, as well as ongoing efforts to develop sophisticated delivery platforms to deliver multiple lineage-directing morphogens (multiple GFs) during bone healing.

Performance and biocompatibility of extremely tough alginate/polyacrylamide hydrogels

Although hydrogels now see widespread use in a host of applications, low fracture toughness and brittleness have limited their more broad use. As a recently described interpenetrating network (IPN) of alginate and polyacrylamide demonstrated a fracture toughness of ≈ 9000 J/m(2), we sought to explore the biocompatibility and maintenance of mechanical properties of these hydrogels in cell culture and in vivo conditions. These hydrogels can sustain a compressive strain of over 90% with minimal loss of Youngs Modulus as well as minimal swelling for up to 50 days of soaking in culture conditions. Mouse mesenchymal stem cells exposed to the IPN gel-conditioned media maintain high viability, and although cells exposed to conditioned media demonstrate slight reductions in proliferation and metabolic activity (WST assay), these effects are abrogated in a dose-dependent manner. Implantation of these IPN hydrogels into subcutaneous tissue of rats for 8 weeks led to mild fibrotic encapsulation and minimal inflammatory response. These results suggest the further exploration of extremely tough alginate/PAAM IPN hydrogels as biomaterials.

Influences of age and mechanical stability on volume, microstructure, and mineralization of the fracture callus during bone healing: Is osteoclast activity the key to age-related impaired healing?

Earlier studies have shown that the influence of fixation stability on bone healing diminishes with advanced age. The goal of this study was to unravel the relationship between mechanical stimulus and age on callus competence at a tissue level. Using 3D in vitro micro-computed tomography derived metrics, 2D in vivo radiography, and histology, we investigated the influences of age and varying fixation stability on callus size, geometry, microstructure, composition, remodeling, and vascularity. Compared were four groups with a 1.5-mm osteotomy gap in the femora of Sprague-Dawley rats: Young rigid (YR), Young semirigid (YSR), Old rigid (OR), Old semirigid (OSR). Hypothesis was that calcified callus microstructure and composition is impaired due to the influence of advanced age, and these individuals would show a reduced response to fixation stabilities. Semirigid fixations resulted in a larger DeltaCSA (Callus cross-sectional area) compared to rigid groups. In vitro microCT analysis at 6 weeks postmortem showed callus bridging scores in younger animals to be superior than their older counterparts (p<0.01). Younger animals showed (i) larger callus strut thickness (p<0.001), (ii) lower perforation in struts (p<0.01), and (iii) higher mineralization of callus struts (p<0.001). Callus mineralization was reduced in young animals with semirigid fracture fixation but remained unaffected in the aged group. While stability had an influence, age showed none on callus size and geometry of callus. With no differences observed in relative osteoid areas in the callus ROI, old as well as semirigid fixated animals showed a higher osteoclast count (p<0.05). Blood vessel density was reduced in animals with semirigid fixation (p<0.05). In conclusion, in vivo monitoring indicated delayed callus maturation in aged individuals. Callus bridging and callus competence (microstructure and mineralization) were impaired in individuals with an advanced age. This matched with increased bone resorption due to higher osteoclast numbers. Varying fixator configurations in older individuals did not alter the dominant effect of advanced age on callus tissue mineralization, unlike in their younger counterparts. Age-associated influences appeared independent from stability. This study illustrates the dominating role of osteoclastic activity in age-related impaired healing, while demonstrating the optimization of fixation parameters such as stiffness appeared to be less effective in influencing healing in aged individuals.

Dose-response relationship of mesenchymal stem cell transplantation and functional regeneration after severe skeletal muscle injury in rats.

Various therapeutic strategies that aim to influence clinical outcome after severe skeletal muscle trauma have been considered. One such method, the local transplantation of stem cells, has been shown to improve tissue regeneration. The number of cells required for successful regeneration, however, remains unclear. The aim of this study was therefore to examine the correlation between the number of transplanted bone marrow-derived mesenchymal stem cells (MSCs) and the resulting muscle function. One week after inducing an open crush trauma in 34 female Sprague Dawley rats, increasing quantities of autologous MSCs (0.1 x 10(6), 1 x 10(6), 2.5 x 10(6), and 10 x 10(6) cells) or saline solution (control group) were transplanted into the left soleus muscle of the rat hind limb. At 4 weeks posttrauma, the outcome was assessed by measuring muscle contraction forces following an indirect fast twitch and tetanic stimulation. A logarithmic dose-response relationship was observed for both maximum twitch and tetanic contraction forces (R(2) = 0.9 for fast twitch [p = 0.004]; R(2) = 0.87 [p = 0.002] for tetanic contraction). The transplantation of 10 x 10(6) cells resulted in the most pronounced improvement of muscle force. MSC therapy represents a promising new tool for the treatment of skeletal muscle trauma that shows potential for aiding in the prevention of severe functional deficiencies. The logarithmic dose-response relationship demonstrates the association between the number of transplanted cells and the resulting muscle forces, as well as the amount of MSCs required for promoting muscular regeneration.

Designing biomimetic scaffolds for bone regeneration: why aim for a copy of mature tissue properties if nature uses a different approach?

This review aims to address the current limitations in biomaterial scaffold-based treatment strategies for bone defect healing and suggests new, alternative approaches that merit further investigation. The question of whether the biomaterial scaffold properties should mimic the natural extracellular matrix of mature tissue or some phase of the dynamic range of tissues observed during the healing process is discussed. Additionally, the authors advocate for a biomimetic approach, which uses the endogenous secondary fracture healing processes to inform the design of scaffold constructs. In particular, the mechanical environment is emphasized as an important factor influencing the clinical success of these constructs. The authors stress the need for a scaffolds design that provides an optimal mechanical environment for cell fate, supplies necessary signals and nutrition to the cells and, thus, more closely mimics the natural healing cascade.

Biphasic Ferrogels for Triggered Drug and Cell Delivery

Ferrogels are an attractive material for many biomedical applications due to their ability to deliver a wide variety of therapeutic drugs on-demand. However, typical ferrogels have yet to be optimized for use in cell-based therapies, as they possess limited ability to harbor and release viable cells. Previously, an active porous scaffold that exhibits large deformations and enhanced biological agent release under moderate magnetic fields has been demonstrated. Unfortunately, at small device sizes optimal for implantation (e.g., 2 mm thickness), these monophasic ferrogels no longer achieve significant deformation due to a reduced body force. A new biphasic ferrogel, containing an iron oxide gradient, capable of large deformations and triggered release even at small gel dimensions, is presented in this study. Biphasic ferrogels demonstrate increased porosity, enhanced mechanical properties, and potentially increased biocompatibility due to their reduced iron oxide content. With their ability to deliver drugs and cells on-demand, it is expected that these ferrogels will have wide utility in the fields of tissue engineering and regenerative medicine.

Presentation of BMP-2 mimicking peptides in 3D hydrogels directs cell fate commitment in osteoblasts and mesenchymal stem cells.

Many strategies for controlling the fate of transplanted stem cells rely on the concurrent delivery of soluble growth factors that have the potential to produce undesirable secondary effects in surrounding tissue. Such off target effects could be eliminated by locally presenting growth factor peptide mimics from biomaterial scaffolds to control stem cell fate. Peptide mimics of bone morphogenetic protein 2 (BMP-2) were synthesized by solid phase Fmoc-peptide synthesis and covalently bound to alginate hydrogels via either carbodiimide or sulfhydryl-based coupling strategies. Successful peptide conjugation was confirmed by (1)H NMR spectroscopy and quantified by fluorescently labeling the peptides. Peptides derived from the knuckle epitope of BMP-2, presented from both 2D surfaces and 3D alginate hydrogels, were shown to increase alkaline phosphatase activity in clonally derived murine osteoblasts. Furthermore, when presented in 3D hydrogels, these peptides were shown to initiate Smad signaling, upregulate osteopontin production, and increase mineral deposition with clonally derived murine mesenchymal stem cells. These data suggest that these peptide-conjugated hydrogels may be effective alternatives to local BMP-2 release in directly and spatially eliciting osteogenesis from transplanted or host osteoprogenitors in the future.

Intra-operatively customized implant coating strategies for local and controlled drug delivery to bone.

Bone is one of the few tissues in the human body with high endogenous healing capacity. However, failure of the healing process presents a significant clinical challenge; it is a tremendous burden for the individual and has related health and economic consequences. To overcome such healing deficits, various concepts for a local drug delivery to bone have been developed during the last decades. However, in many cases these concepts do not meet the specific requirements of either surgeons who must use these strategies or individual patients who might benefit from them. We describe currently available methods for local drug delivery and their limitations in therapy. Various solutions for drug delivery to bone focusing on clinical applications and intra-operative constraints are discussed and drug delivery by implant coating is highlighted. Finally, a new set of design and performance requirements for intra-operatively customized implant coatings for controlled drug delivery is proposed. In the future, these requirements may improve approaches for local and intra-operative treatment of patients.

A new device to control mechanical environment in bone defect healing in rats

Mechanical conditions have a significant influence on the biological processes of bone healing. Small animal models that allow controlling the mechanical environment of fracture and bone defect healing are needed. The aim of this study was to develop a new animal model that allows to reliably control the mechanical environment in fracture and bone defect healing in rats using different implant materials. An external fixator was designed and mounted in vitro to rat femurs using four Kirschner-wires (titanium (T) or steel (S)) of 1.2 mm diameter. The specimens were distracted to a gap of 1.5mm. Axial and torsional stiffness of the device was tested increasing the offset (distance between bone and fixator crossbar) from 5 to 15 mm. In vivo performance (well-being, infection, breaking of wires and bone healing) was evaluated in four groups of 24 Sprague-Dawley rats varying in offset (7.5 and 15 mm) and implant material (S/T) over 6 weeks. Torsional and axial stiffness were higher in steel compared to titanium setups. A decrease in all configurations was observed by increasing the offset. The offset 7.5 mm showed a significantly higher torsional (S: p<0.01, T: p<0.001) and axial in vitro stiffness (S: p<0.001, T: p<0.001) compared to 15 mm offset of the fixator. Although in vitro designed to be different in mechanical stiffness, no difference was found between the groups regarding complication rate. The overall-complication rate was 5.2%. In conclusion, we were able to establish a small animal model for bone defect healing which allows modeling the mechanical conditions at the defect site in a defined manner.