Ben P. Hung

Stromal cells and stem cells in clinical bone regeneration

Stem-cell-mediated bone repair has been used in clinical trials for the regeneration of large craniomaxillofacial defects, to slow the process of bone degeneration in patients with osteonecrosis of the femoral head and for prophylactic treatment of distal tibial fractures. Successful regenerative outcomes in these investigations have provided a solid foundation for wider use of stromal cells in skeletal repair therapy. However, employing stromal cells to facilitate or enhance bone repair is far from being adopted into clinical practice. Scientific, technical, practical and regulatory obstacles prevent the widespread therapeutic use of stromal cells. Ironically, one of the major challenges lies in the limited understanding of the mechanisms via which transplanted cells mediate regeneration. Animal models have been used to provide insight, but these models largely fail to reproduce the nuances of human diseases and bone defects. Consequently, the development of targeted approaches to optimize cell-mediated outcomes is difficult. In this Review, we highlight the successes and challenges reported in several clinical trials that involved the use of bone-marrow-derived mesenchymal or adipose-tissue-derived stromal cells. We identify several obstacles blocking the mainstream use of stromal cells to enhance skeletal repair and highlight technological innovations or areas in which novel techniques might be particularly fruitful in continuing to advance the field of skeletal regenerative medicine.

Engineering anatomically shaped vascularized bone grafts with hASCs and 3D‐printed PCL scaffolds

The treatment of large craniomaxillofacial bone defects is clinically challenging due to the limited availability of transplantable autologous bone grafts and the complex geometry of the bones. The ability to regenerate new bone tissues that faithfully replicate the anatomy would revolutionize treatment options. Advances in the field of bone tissue engineering over the past few decades offer promising new treatment alternatives using biocompatible scaffold materials and autologous cells. This approach combined with recent advances in three-dimensional (3D) printing technologies may soon allow the generation of large, bioartificial bone grafts with custom, patient-specific architecture. In this study, we use a custom-built 3D printer to develop anatomically shaped polycaprolactone (PCL) scaffolds with varying internal porosities. These scaffolds are assessed for their ability to support induction of human adipose-derived stem cells (hASCs) to form vasculature and bone, two essential components of functional bone tissue. The development of functional tissues is assessed in vitro and in vivo. Finally, we demonstrate the ability to print large mandibular and maxillary bone scaffolds that replicate fine details extracted from patients computed tomography scans. The findings of this study illustrate the capabilities and potential of 3D printed scaffolds to be used for engineering autologous, anatomically shaped, vascularized bone grafts.

Cystamine-terminated poly(beta-amino ester)s for siRNA delivery to human mesenchymal stem cells and enhancement of osteogenic differentiation

Enhancing human mesenchymal stem cell (hMSC) differentiation via RNA interference (RNAi) could provide an effective way of controlling cell fate for tissue engineering, but a safe and effective delivery vehicle must first be developed. Here, we evaluated an array of synthetic end-modified poly(beta-amino ester) (PBAE)-based nanoparticles to optimize siRNA delivery into hMSCs. In general, cystamine-terminated polymers caused the most knockdown, with the best polymer achieving 91% knockdown 20 days post-transfection. Binding studies revealed that the cystamine-terminated polymer bound siRNA tightly at lower weight ratios of polymer to siRNA but then efficiently released siRNA upon exposure to a reducing environment, suggesting that this class of PBAEs can form tight initial interactions with its cargo and then cause efficient, environmentally-triggered release in the cytoplasm. Finally, we tested a functional application of this system by transfecting hMSCs with siRNA against an inhibitor of osteogenesis, B-cell lymphoma (Bcl)-like protein 2 (BCL2L2). This resulted in enhanced osteogenesis over 4 weeks as evidenced by Alizarin Red S staining and calcium quantification. The bioreducible PBAE/siRNA nanoparticles developed here can provide a means of safe and effective control of hMSC differentiation for a wide variety of applications.

Mechanical control of tissue-engineered bone

Bone is a load-bearing tissue and physical forces play key roles in the development and maintenance of its structure. Mechanical cues can stimulate the expression of an osteogenic phenotype, enhance matrix and mineral deposition, and influence tissue organization to improve the functional outcome of engineered bone grafts. In recent years, a number of studies have investigated the effects of biophysical forces on the bone formation properties of osteoprogenitor cells. The application of physiologically relevant stimuli to tissue-engineered bone may be determined through observation and understanding of forces to which osteoblasts, osteoclasts, and osteocytes are exposed in native bone. Subsequently, these cues may be parameterized and their effects studied in well-defined in vitro systems. The osteo-inductive effects of three specific mechanical cues - shear stress, substrate rigidity, and nanotopography - on cells cultured in monolayer or in three-dimensional biomaterial scaffolds in vitro are reviewed. Additionally, we address the time-dependent effects of mechanical cues on vascular infiltration and de novo bone formation in acellular scaffolds implanted into load-bearing sites in vivo. Recent studies employing cutting-edge advances in biomaterial fabrication and bioreactor design have provided key insights into the role of mechanical cues on cellular fate and tissue properties of engineered bone grafts. By providing mechanistic understanding, future studies may go beyond empirical approaches to rational design of engineering systems to control tissue development.

3D-Printing Technologies for Craniofacial Rehabilitation, Reconstruction, and Regeneration

The treatment of craniofacial defects can present many challenges due to the variety of tissue-specific requirements and the complexity of anatomical structures in that region. 3D-printing technologies provide clinicians, engineers and scientists with the ability to create patient-specific solutions for craniofacial defects. Currently, there are three key strategies that utilize these technologies to restore both appearance and function to patients: rehabilitation, reconstruction and regeneration. In rehabilitation, 3D-printing can be used to create prostheses to replace or cover damaged tissues. Reconstruction, through plastic surgery, can also leverage 3D-printing technologies to create custom cutting guides, fixation devices, practice models and implanted medical devices to improve patient outcomes. Regeneration of tissue attempts to replace defects with biological materials. 3D-printing can be used to create either scaffolds or living, cellular constructs to signal tissue-forming cells to regenerate defect regions. By integrating these three approaches, 3D-printing technologies afford the opportunity to develop personalized treatment plans and design-driven manufacturing solutions to improve aesthetic and functional outcomes for patients with craniofacial defects.

Platelet‐Derived Growth Factor BB Enhances Osteogenesis of Adipose‐Derived But Not Bone Marrow‐Derived Mesenchymal Stromal/Stem Cells

Tissue engineering using mesenchymal stem cells (MSCs) holds great promise for regenerating critically sized bone defects. While the bone marrow‐derived MSC is the most widely studied stromal/stem cell type for this application, its rarity within bone marrow and painful isolation procedure have motivated investigation of alternative cell sources. Adipose‐derived stromal/stem cells (ASCs) are more abundant and more easily procured; furthermore, they also possess robust osteogenic potency. While these two cell types are widely considered very similar, there is a growing appreciation of possible innate differences in their biology and response to growth factors. In particular, reports indicate that their osteogenic response to platelet‐derived growth factor BB (PDGF‐BB) is markedly different: MSCs responded negatively or not at all to PDGF‐BB while ASCs exhibited enhanced mineralization in response to physiological concentrations of PDGF‐BB. In this study, we directly tested whether a fundamental difference existed between the osteogenic responses of MSCs and ASCs to PDGF‐BB. MSCs and ASCs cultured under identical osteogenic conditions responded disparately to 20 ng/ml of PDGF‐BB: MSCs exhibited no difference in mineralization while ASCs produced more calcium per cell. siRNA‐mediated knockdown of PDGFRβ within ASCs abolished their ability to respond to PDGF‐BB. Gene expression was also different; MSCs generally downregulated and ASCs generally upregulated osteogenic genes in response to PDGF‐BB. ASCs transduced to produce PDGF‐BB resulted in more regenerated bone within a critically sized murine calvarial defect compared to control ASCs, indicating PDGF‐BB used specifically in conjunction with ASCs might enhance tissue engineering approaches for bone regeneration. Stem Cells 2015;33:2773–2784

Tumor Necrosis Factor Improves Vascularization in Osteogenic Grafts Engineered with Human Adipose-Derived Stem/Stromal Cells

The innate immune response following bone injury plays an important role in promoting cellular recruitment, revascularization, and other repair mechanisms. Tumor necrosis factor-α (TNF) is a prominent pro-inflammatory cytokine in this cascade, and has been previously shown to improve bone formation and angiogenesis in a dose- and timing-dependent manner. This ability to positively impact both osteogenesis and vascular growth may benefit bone tissue engineering, as vasculature is essential to maintaining cell viability in large grafts after implantation. Here, we investigated the effects of exogenous TNF on the induction of adipose-derived stem/stromal cells (ASCs) to engineer pre-vascularized osteogenic tissue in vitro with respect to dose, timing, and co-stimulation with other inflammatory mediators. We found that acute (2-day), low-dose exposure to TNF promoted vascularization, whereas higher doses and continuous exposure inhibited vascular growth. Co-stimulation with platelet-derived growth factor (PDGF), another key factor released following bone injury, increased vascular network formation synergistically with TNF. ASC-seeded grafts were then cultured within polycaprolactone-fibrin composite scaffolds and implanted in nude rats for 2 weeks, resulting in further tissue maturation and increased angiogenic ingrowth in TNF-treated grafts. VEGF-A expression levels were significantly higher in TNF-treated grafts immediately prior to implantation, indicating a long-term pro-angiogenic effect. These findings demonstrate that TNF has the potential to promote vasculogenesis in engineered osteogenic grafts both in vitro and in vivo. Thus, modulation and/or recapitulation of the immune response following bone injury may be a beneficial strategy for bone tissue engineering.

Engineering Bone Grafts with Enhanced Bone Marrow and Native Scaffolds

The translation of tissue engineering approaches to the clinic has been hampered by the inability to find suitable multipotent cell sources requiring minimal in vitro expansion. Enhanced bone marrow (eBM), which is obtained by reaming long bone medullary canals and isolating the solid marrow putty, has large quantities of stem cells and demonstrates significant potential to regenerate bone tissues. eBM, however, cannot impart immediate load-bearing mechanical integrity or maintain the gross anatomical structure to guide bone healing. Yet, its putty-like consistency creates a challenge for obtaining the uniform seeding necessary to effectively combine it with porous scaffolds. In this study, we examined the potential for combining eBM with mechanically strong, osteoinductive trabecular bone scaffolds for bone regeneration by creating channels into scaffolds for seeding the eBM. eBM was extracted from the femurs of adult Yorkshire pigs using a Synthes reamer-irrigator-aspirator device, analyzed histologically, and digested to extract cells and characterize their differentiation potential. To evaluate bone tissue formation, eBM was seeded into the channels in collagen-coated or noncoated scaffolds, cultured in osteogenic conditions for 4 weeks, harvested and assessed for tissue distribution and bone formation. Our data demonstrates that eBM is a heterogenous tissue containing multipotent cell populations. Furthermore, coating scaffolds with a collagen hydrogel significantly enhanced cellular migration, promoted uniform tissue development and increased bone mineral deposition. These findings suggest the potential for generating customized autologous bone grafts for treating critical-sized bone defects by combining a readily available eBM cell source with decellularized trabecular bone scaffolds.

Bioreactor Technology for Oral and Craniofacial Tissue Engineering

Abstract Tissue engineering approaches hold great promise for the remediation of craniofacial defects to various tissues including bone, cartilage, ligaments, meniscus, blood vessels, and nerves. These strategies include in vitro cultivation of cells with biomaterials to achieve construct maturation prior to implantation into the defect site. However, culturing large, viable three-dimensional constructs is an engineering challenge, since the viability of cells within the construct is heavily dependent on adequate oxygen and nutrient delivery. To prevent the formation of necrotic cores and facilitate uniform tissue growth within the graft, bioreactors have been used to provide convective transfer. Additionally, bioreactors may be used to mimic the physiological stresses imparted to native tissues, which stimulate their functional organization. This chapter discusses the rationale for using bioreactors, the principles underlying various bioreactor designs, and provides case studies to demonstrate how the use of custom-designed bioreactors enhances the functional outcome of engineered tissues.

Chapter 10 – Craniofacial Bone

Craniofacial bone loss caused by trauma, cancer resection, or congenital diseases significantly impacts patients’ facial appearance. An estimated 20% of annual bone transplants in the United States occur in the craniofacial region. Tissue engineering approaches that combine cells, scaffolds, and stimulatory cues hold considerable potential for regenerating functional bone. Several modalities are suited to generating scaffolds with the complex anatomical geometries of the craniofacial bones. Of these, three-dimensional printing (3DP) is particularly attractive as it is capable of providing precise control over both the macroscopic and microscopic architecture of the scaffold. In this chapter, we review various 3DP methods and their application in craniofacial bone tissue engineering. In addition, we examine how these techniques may be combined with nanocomposites to mimic bone composition and structure, and deliver bioactive molecules for regeneration and reconstruction of craniofacial bone.