Adipogenic differentiation of hematopoietic lineage cells isolated from adipose tissue of humans.

We previously discovered some adipocytes in the major white fat depots of mice and humans arise from bone marrow-derived cells of hematopoietic lineage rather than conventional mesenchymal precursors, termed bone marrow-derived adipocytes (BMDA). Here we aimed to determine if hematopoietic lineage cells isolated from adipose tissue and circulation of humans could undergo adipogenic differentiation in vitro, thereby establishing an in vitro model for studies of BMDA. We hypothesized that hematopoietic lineage cells isolated from adipose tissue, but not circulation, of humans would demonstrate adipogenic potential. Participants included younger (20-50 years) and older (>50-75 years) men and women, BMI 20-37 kg/m2. Subcutaneous abdominal adipose tissue biopsies were obtained and stromal cell populations identified by flow cytometry. Sorted cells underwent in vitro cultivation via traditional mesenchymal culture methodology (mesenchymal lineage) or a novel 3D-fibrin clot followed by traditional adherent culture (hematopoietic lineage) for assessment of proliferation and differentiation capacity. We found hematopoietic lineage cells isolated from the adipose tissue stroma, but not the circulation, were capable of proliferation and multilineage (adipogenic and osteogenic) differentiation in vitro. We provide a new investigative tool that can be used to perform translational studies of BMDAs and provide initial evidence that hematopoietic lineage cells isolated from the adipose tissue of humans can undergo hematopoietic-to-mesenchymal transition with multilineage differentiation potential in an in vitro environment.

Hydroxyapatite-coated gelatin microribbon scaffolds induce rapid endogenous cranial bone regeneration in vivo.

Hydroxyapatite (HA) has a composition similar to mineral bone and has been used for coating macroporous scaffolds to enhance bone formation. However, previous macroporous scaffolds did not support minimally invasive delivery. Our lab has reported on gelatin-based microribbon (µRB) shaped hydrogels, which combine injectability with macroporosity and support cranial bone formation in an immunocompromised mouse model. However, gelatin alone was not sufficient to support cranial bone formation in immunocompetent animals. To overcome this challenge, here we evaluated two methods to incorporate HA into gelatin µRB scaffolds using either modified simulated body fluid (mSBF) or commercially available HA nanoparticles (HAnp). HA incorporation and distribution were characterized using scanning electron microscopy and energy-dispersive X-ray spectroscopy. While both methods enhanced MSC osteogenesis and mineralization, the mSBF method led to undesirable reduction in mechanical properties. HAnp-coated µRB scaffolds were further evaluated in an immunocompetent mouse cranial defect model. Acellular HAnp-coated gelatin µRB scaffolds induced rapid and robust endogenous cranial bone regeneration as shown by MicroCT imaging and histology. Co-delivery with exogenous MSCs led to later bone resorption accompanied by increased osteoclast activity. In summary, our results demonstrate the promise of gelatin µRBs with HAnps as a promising therapy for cranial bone regeneration without the need for exogenous cells or growth factors.

Injectable and in situ crosslinkable gelatin microribbon hydrogels for stem cell delivery and bone regeneration in vivo.

Rationale: Injectable matrices are highly desirable for stem cell delivery. Previous research has highlighted the benefit of scaffold macroporosity in enhancing stem cell survival and bone regeneration in vivo. However, there remains a lack of injectable and in situ crosslinkable macroporous matrices for stem cell delivery to achieve fast bone regeneration in immunocompetent animal model. The goal of this study is to develop an injectable gelatin-based µRB hydrogel supporting direct cell encapsulation that is available in clinics as macroporous matrices to enhance adipose-derived stromal cell (ASC) survival, engraftment and accelerate bone formation in craniofacial defect mouse. Methods: Injectable and in situ crosslinkable gelatin microribbon (µRB)-based macroporous hydrogels were developed by wet-spinning. Injectability was optimized by varying concentration of glutaraldehyde for intracrosslinking of µRB shape, and fibrinogen coating. The efficacy of injectable µRBs to support ASCs delivery and bone regeneration were further assessed in vivo using an immunocompetent mouse cranial defect model. ASCs survival was evaluated by bioluminescent imaging and bone regeneration was assessed by micro-CT. The degradation and biocompatibility were determined by histological analysis. Results: We first optimized injectability by varying concentration of glutaraldehyde used to fix gelatin µRBs. The injectable µRB formulation were subsequently coated with fibrinogen, which allows in situ crosslinking by thrombin. Fluorescence imaging and histology showed majority of µRBs degraded by the end of 3 weeks. Injectable µRBs supported comparable level of ASC proliferation and bone regeneration as implantable prefabricated µRB controls. Adding low dosage of BMP2 (100 ng per scaffold) with ASCs substantially accelerated the speed of mineralized bone regeneration, with 90% of the bone defect refilled by week 8. Immunostaining showed M1 (pro-inflammatory) macrophages were recruited to the defect at day 3, and was replaced by M2 (anti-inflammatory) macrophages by week 2. Adding µRBs or BMP2 did not alter macrophage response. Injectable µRBs supported vascularization, and BMP-2 further enhanced vascularization. Conclusions: Our results demonstrated that µRB-based scaffolds enhanced ASC survival and accelerated bone regeneration after injection into critical sized cranial defect mouse. Such injectable µRB-based scaffold can provide a versatile biomaterial for delivering various stem cell types and enhancing tissue regeneration.

Interleukin-4 overexpressing mesenchymal stem cells within gelatin-based microribbon hydrogels enhance bone healing in a murine long bone critical-size defect model.

Mesenchymal stem cell (MSC)-based therapy is a promising strategy for bone repair. Furthermore, the innate immune system, and specifically macrophages, plays a crucial role in the differentiation and activation of MSCs. The anti-inflammatory cytokine Interleukin-4 (IL-4) converts pro-inflammatory M1 macrophages into a tissue regenerative M2 phenotype, which enhances MSC differentiation and function. We developed lentivirus-transduced IL-4 overexpressing MSCs (IL-4 MSCs) that continuously produce IL-4 and polarize macrophages toward an M2 phenotype. In the current study, we investigated the potential of IL-4 MSCs delivered using a macroporous gelatin-based microribbon (µRB) scaffold for healing of critical-size long bone defects in Mice. IL-4 MSCs within µRBs enhanced M2 marker expression without inhibiting M1 marker expression in the early phase, and increased macrophage migration into the scaffold. Six weeks after establishing the bone defect, IL-4 MSCs within µRBs enhanced bone formation and helped bridge the long bone defect. IL-4 MSCs delivered using macroporous µRB scaffold is potentially a valuable strategy for the treatment of critical-size long bone defects.

Gelatin-Based Microribbon Hydrogels Support Robust MSC Osteogenesis across a Broad Range of Stiffness.

Scaffold macroporosity has been shown to be critical for promoting bone regeneration. Although injectable materials are preferred for minimally invasive delivery, conventional macroporous scaffolds were not injectable and do not support homogeneous cell encapsulation. We recently reported a gelatin-based microribbon (µRB) scaffold that offers macroporosity while also supporting homogeneous cell encapsulation. Compared to conventional gelatin hydrogels, macroporous gelatin µRB scaffolds demonstrated great advantage in enhancing mesenchymal stem cell (MSC)-based cartilage formation. However, whether gelatin-based µRBs support MSC osteogenesis and bone formation remains unknown. The goal of this study is to assess the potential of gelatin-based µRBs for supporting MSC-based osteogenesis and bone formation in vitro. Given recent evidence from the literature that osteogenesis is sensitive to substrate stiffness, we further investigate how varying µRB stiffness modulates MSC osteogenesis. We first determine the maximal stiffness range of gelatin µRBs that can be fabricated (13-57 kPa), which supports both retention of µRB shape and macroporosity within scaffolds after inter-cross-linking. Interestingly, varying µRB stiffness across a broad range of stiffness did not significantly impact osteogenesis, with all groups supporting upregulation of bone markers and extensive collagen deposition. All gelatin µRBs also supported a comparable level of cell spreading and upregulation of mechanosensing markers. However, soft µRB (13 kPa) scaffolds did not maintain structural integrity and condensed into a pellet over time. Both intermediate and stiff gelatin µRB-based scaffolds maintained their integrity and supported robust bone formation, leading to a more than 10-fold increase in the compressive moduli of engineered bone after 5 weeks of culture in osteogenic media. Incorporating hydroxyapatite (HA) nanoparticle coating onto the gelatin µRB surface further accelerated the maturation of MSCs into osteoblasts and mineralization. Together, these results validate that gelatin µRBs can support MSC osteogenesis across a broad range of stiffness and offers an injectable macroporous scaffold for enhancing stem-cell-based bone regeneration.

Gelatin-Based Microribbon Hydrogels Accelerate Cartilage Formation by Mesenchymal Stem Cells in Three Dimensions.

Hydrogels (HGs) are attractive matrices for cell-based cartilage tissue regeneration given their injectability and ability to fill defects with irregular shapes. However, most HGs developed to date often lack cell scale macroporosity, which restrains the encapsulated cells, leading to delayed new extracellular matrix deposition restricted to pericellular regions. Furthermore, tissue-engineered cartilage using conventional HGs generally suffers from poor mechanical property and fails to restore the load-bearing property of articular cartilage. The goal of this study was to evaluate the potential of macroporous gelatin-based microribbon (µRB) HGs as novel 3D matrices for accelerating chondrogenesis and new cartilage formation by human mesenchymal stem cells (MSCs) in 3D with improved mechanical properties. Unlike conventional HGs, these µRB HGs are inherently macroporous and exhibit cartilage-mimicking shock-absorbing mechanical property. After 21 days of culture, MSC-seeded µRB scaffolds exhibit a 20-fold increase in compressive modulus to 225 kPa, a range that is approaching the level of native cartilage. In contrast, HGs only resulted in a modest increase in compressive modulus of 65 kPa. Compared with conventional HGs, macroporous µRB scaffolds significantly increased the total amount of neocartilage produced by MSCs in 3D, with improved interconnectivity and mechanical strength. Altogether, these results validate gelatin-based µRBs as promising scaffolds for enhancing and accelerating MSC-based cartilage regeneration and may be used to enhance cartilage regeneration using other cell types as well.

Winner of the Young Investigator Award of the Society for Biomaterials at the 10th World Biomaterials Congress, May 17-22, 2016, Montreal QC, Canada: Microribbon-based hydrogels accelerate stem cell-based bone regeneration in a mouse critical-size cranial defect model.

Stem cell-based therapies hold great promise for enhancing tissue regeneration. However, the majority of cells die shortly after transplantation, which greatly diminishes the efficacy of stem cell-based therapies. Poor cell engraftment and survival remain a major bottleneck to fully exploiting the power of stem cells for regenerative medicine. Biomaterials such as hydrogels can serve as artificial matrices to protect cells during delivery and guide desirable cell fates. However, conventional hydrogels often lack macroporosity, which restricts cell proliferation and delays matrix deposition. Here we report the use of injectable, macroporous microribbon (µRB) hydrogels as stem cell carriers for bone repair, which supports direct cell encapsulation into a macroporous scaffold with rapid spreading. When transplanted in a critical-sized, mouse cranial defect model, µRB-based hydrogels significantly enhanced the survival of transplanted adipose-derived stromal cells (ADSCs) (81%) and enabled up to three-fold cell proliferation after 7 days. In contrast, conventional hydrogels only led to 27% cell survival, which continued to decrease over time. MicroCT imaging showed µRBs enhanced and accelerated mineralized bone repair compared to hydrogels (61% vs. 34% by week 6), and stem cells were required for bone repair to occur. These results suggest that paracrine signaling of transplanted stem cells are responsible for the observed bone repair, and enhancing cell survival and proliferation using µRBs further promoted the paracrine-signaling effects of ADSCs for stimulating endogenous bone repair. We envision µRB-based scaffolds can be broadly useful as a novel scaffold for enhancing stem cell survival and regeneration of other tissue types. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 1321-1331, 2016.