Michael D. Hoffman

Degradable hydrogels for spatiotemporal control of mesenchymal stem cells localized at decellularized bone allografts

The transplantation of cells, such as mesenchymal stem cells (MSCs), has numerous applications in the field of regenerative medicine. For cell transplantation strategies to be successful therapeutically, cellular localization and persistence must be controlled to maximize cell-mediated contributions to healing. Herein, we demonstrate that hydrolytic degradation of poly(ethylene glycol) (PEG) hydrogels can be used to spatiotemporally control encapsulated MSC localization to decellularized bone allografts, both in vitro and in vivo. By altering the number of hydrolytically degradable lactide repeat units within PEG-d,l-lactide-methacrylate macromers, a series of hydrogels was synthesized that degraded over ∼1, 2 and 3weeks. MSCs were encapsulated within these hydrogels formed around decellularized bone allografts, and non-invasive, longitudinal fluorescence imaging was used to track cell persistence both in vitro and in vivo. Spatiotemporal localization of MSCs to the exterior of bone allograft surfaces was similar to in vitro hydrogel degradation kinetics despite hydrogel mesh sizes being ∼2-3 orders of magnitude smaller than MSC size throughout the degradation process. Thus, localized, cell-mediated degradation and MSC migration from the hydrogels are suspected, particularly as ∼10% of the total transplanted MSC population was shown to persist in close proximity (within ∼650μm) to grafts 7weeks after complete hydrogel degradation. This work demonstrates the therapeutic utility of PEG-based hydrogels for controlling spatiotemporal cell transplantation for a myriad of regenerative medicine strategies.

Agonism of Wnt-β-catenin signalling promotes mesenchymal stem cell (MSC) expansion.

Promoting mesenchymal stem cell (MSC) proliferation has numerous applications in stem cell therapies, particularly in the area of regenerative medicine. In order for cell‐based regenerative approaches to be realized, MSC proliferation must be achieved in a controlled manner without compromising stem cell differentiation capacities. Here we demonstrate that 6‐bromoindirubin‐3′‐oxime (BIO) increases MSC β‐catenin activity 106‐fold and stem cell‐associated gene expression ~33‐fold, respectively, over untreated controls. Subsequently, BIO treatment increases MSC populations 1.8‐fold in typical 2D culture conditions, as well as 1.3‐fold when encapsulated within hydrogels compared to untreated cells. Furthermore, we demonstrate that BIO treatment does not reduce MSC multipotency where MSCs maintain their ability to differentiate into osteoblasts, chondrocytes and adipocytes using standard conditions. Taken together, our results demonstrate BIOs potential utility as a proliferative agent for cell transplantation and tissue regeneration. Copyright

Emulating native periosteum cell population and subsequent paracrine factor production to promote tissue engineered periosteum-mediated allograft healing.

Emulating autograft healing within the context of decellularized bone allografts has immediate clinical applications in the treatment of critical-sized bone defects. The periosteum, a thin, osteogenic tissue that surrounds bone, houses a heterogenous population of stem cells and osteoprogenitors. There is evidence that periosteum-cell derived paracrine factors, specifically vascular endothelial growth factor (VEGF) and bone morphogenetic protein 2 (BMP2), orchestrate autograft healing through host cell recruitment and subsequent tissue elaboration. In previous work, we demonstrated that the use of poly(ethylene glycol) (PEG) hydrogels as a tissue engineered (T.E.) periosteum to localize mesenchymal stem cells (MSCs) to the surface of decellularized bone enhances allograft healing and integration. Herein, we utilize a mixed population of 50:50 MSCs and osteoprogenitor cells to better mimic native periosteum cell population and paracrine factor production to further promote allograft healing. This mixed cell population was localized to the surface of decellularized allografts within degradable hydrogels and shown to expedite allograft healing. Specifically, bone callus formation and biomechanical graft-host integration are increased as compared to unmodified allografts. These results demonstrate the dual importance of periosteum-mediated paracrine factors orchestrating host cell recruitment as well as new bone formation while developing clinically translatable strategies for allograft healing and integration.

Non-contact scanning diffuse correlation tomography system for three-dimensional blood flow imaging in a murine bone graft model.

A non-contact galvanometer-based optical scanning system for diffuse correlation tomography was developed for monitoring bone graft healing in a murine femur model. A linear image reconstruction algorithm for diffuse correlation tomography was tested using finite-element method based simulated data and experimental data from a femur or a tube suspended in a homogeneous liquid phantom. Finally, the non-contact system was utilized to monitor in vivo blood flow changes prior to and one week after bone graft transplantation within murine femurs. Localized blood flow changes were observed in three mice, demonstrating a potential for quantification of longitudinal blood flow associated with bone graft healing.

Non-Invasive Monitoring of Temporal and Spatial Blood Flow during Bone Graft Healing Using Diffuse Correlation Spectroscopy

Vascular infiltration and associated alterations in microvascular blood flow are critical for complete bone graft healing. Therefore, real-time, longitudinal measurement of blood flow has the potential to successfully predict graft healing outcomes. Herein, we non-invasively measure longitudinal blood flow changes in bone autografts and allografts using diffuse correlation spectroscopy in a murine femoral segmental defect model. Blood flow was measured at several positions proximal and distal to the graft site before implantation and every week post-implantation for a total of 9 weeks (autograft n = 7 and allograft n = 10). Measurements of the ipsilateral leg with the graft were compared with those of the intact contralateral control leg. Both autografts and allografts exhibited an initial increase in blood flow followed by a gradual return to baseline levels. Blood flow elevation lasted up to 2 weeks in autografts, but this duration varied from 2 to 6 weeks in allografts depending on the spatial location of the measurement. Intact contralateral control leg blood flow remained at baseline levels throughout the 9 weeks in the autograft group; however, in the allograft group, blood flow followed a similar trend to the graft leg. Blood flow difference between the graft and contralateral legs (ΔrBF), a parameter defined to estimate graft-specific changes, was elevated at 1–2 weeks for the autograft group, and at 2–4 weeks for the allograft group at the proximal and the central locations. However, distal to the graft, the allograft group exhibited significantly greater ΔrBF than the autograft group at 3 weeks post-surgery (p < 0.05). These spatial and temporal differences in blood flow supports established trends of delayed healing in allografts versus autografts.

Disruption of Cell-Cell Contact-mediated Notch Signaling via Hydrogel Encapsulation Reduces Mesenchymal Stem Cell Chondrogenic Potential

Cell-cell contact-mediated Notch signaling is essential for mesenchymal stem cell (MSC) chondrogenesis during development. However, subsequent deactivation of Notch signaling is also required to allow for stem cell chondrogenic progression. Recent literature has shown that Notch signaling can also influence Wnt/β-catenin signaling, critical for MSC differentiation, through perturbations in cell-cell contacts. Traditionally, abundant cell-cell contacts, consistent with development, are emulated in vitro using pellet cultures for chondrogenesis. However, cells are often encapsulated within biomaterials-based scaffolds, such as hydrogels, to improve therapeutic cell localization in vivo. To explore the role of Notch and Wnt/β-catenin signaling in the context of hydrogel-encapsulated MSC chondrogenesis, we compared signaling and differentiation capacity of MSCs in both hydrogels and traditional pellet cultures. We demonstrate that encapsulation within poly(ethylene glycol) hydrogels reduces cell-cell contacts, and both Notch (7.5-fold) and Wnt/β-catenin (84.7-fold) pathway activation. Finally, we demonstrate that following establishment of cell-cell contacts and transient Notch signaling in pellet cultures, followed by Notch signaling deactivation, resulted in a 1.5-fold increase in MSC chondrogenesis. Taken together, these findings support that cellular condensation, and establishment of initial cell-cell contacts is critical for MSC chondrogenesis, and this process is inhibited by hydrogel encapsulation.

3D printed calcium phosphate and poly(caprolactone) composites with improved mechanical properties and preserved microstructure

Biphasic calcium phosphate scaffolds formed via three dimensional (3D) printing technology to exhibit porosity and chemical resorbability to promote osseointegration often lack the strength and toughness required to withstand loading in bone tissue engineering applications. Herein, sintering and CaP:poly(caprolactone) (PCL) composite formation were explored to improve 3D printed scaffold strength and toughness. Hydroxyapatite and α-tricalcium phosphate (α-TCP) biphasic calcium powders were printed using phosphoric acid binder, which generated monetite and hydroxyapatite scaffolds. Upon sintering, evolution of β-TCP was observed along with an increase in flexural strength and modulus but no effect on fracture toughness was observed. Furthermore, scaffold porosity increased with sintering. Additionally, two techniques of PCL composite formation were employed: postprint precipitation and 3D print codeposition to further augment scaffold mechanical properties. While both techniques significantly improved flexural strength, flexural modulus, and fracture toughness under most conditions explored, precipitation yielded more substantial increases in these properties, which is attributed to better continuity of the PCL phase. However, precipitation also compromised surface porosity due to PCL passivation of the calcium phosphate surface, which may subsequently hinder scaffold integration and bone regeneration.

Monitoring blood flow changes induced by nicotinamide injection in mouse leg using diffuse correlation spectroscopy

Diffuse Correlation Spectroscopy was employed to monitor relative blood flow in mouse leg before and after injection of nicotinamide (1000 mg/kg). The result shows an average increase of 172% in relative blood flow, 30 minutes after injection.