Development and systematic characterization of GelMA/alginate/PEGDMA/xanthan gum hydrogel bioink system for extrusion bioprinting.

Gelatin methacryloyl (GelMA)/alginate-based hydrogels have shown great promise in bioprinting, but their printability is limited at room temperature. In this paper, we present our development of a room temperature printable hydrogel bioink by introducing polyethylene glycol dimethacrylate (PEGDMA) and xanthan gum into the GelMA/alginate system. The inclusion of PEGDMA facilitates tuning of the hydrogel's mechanical property, while xanthan gum improves the viscosity of the hydrogel system and allows easy extrusion at room temperature. To fine-tune the mechanical and degradation properties, methacrylated xanthan gum was synthesized and chemically crosslinked to the system. We systematically characterized this hydrogel with attention to printability, strut size, mechanical property, degradation and cytocompatibility, and achieved a broad range of compression modulus (∼10-100 kPa) and degradation profile (100% degradation by 24 h-40% by 2 weeks). Moreover, xanthan gum demonstrated solubility in ionic solutions such as cell culture medium, which is essential for biocompatibility. Live/dead staining showed that cell viability in the printed hydrogels was over 90% for 7 days. Metabolic activity analysis demonstrated excellent cell proliferation and survival within 4 weeks of incubation. In summary, the newly developed hydrogel system has demonstrated distinct features including extrusion printability, widely tunable mechanical property and degradation, ionic solubility, and cytocompatibility. It offers great flexibility in bioprinting and tissue engineering.

Hybprinting for musculoskeletal tissue engineering.

This review presents bioprinting methods, biomaterials, and printing strategies that may be used for composite tissue constructs for musculoskeletal applications. The printing methods discussed include those that are suitable for acellular and cellular components, and the biomaterials include soft and rigid components that are suitable for soft and/or hard tissues. We also present strategies that focus on the integration of cell-laden soft and acellular rigid components under a single printing platform. Given the structural and functional complexity of native musculoskeletal tissue, we envision that hybrid bioprinting, referred to as hybprinting, could provide unprecedented potential by combining different materials and bioprinting techniques to engineer and assemble modular tissues.

A bioactive synthetic membrane improves bone healing in a preclinical nonunion model.

OBJECTIVES: High energy long bone fractures with critical bone loss are at risk for nonunion without strategic intervention. We hypothesize that a synthetic membrane implanted at a single stage improves bone healing in a preclinical nonunion model. METHODS: Using standard laboratory techniques, microspheres encapsulating bone morphogenic protein-2 (BMP2) or platelet derived growth factor (PDGF) were designed and coupled to a type 1 collagen sheet. Critical femoral defects were created in rats and stabilized by locked retrograde intramedullary nailing. The negative control group had an empty defect. The induced membrane group (positive control) had a polymethylmethacrylate spacer inserted into the defect for four weeks and replaced with a bare polycaprolactone/beta-tricalcium phosphate (PCL/ß-TCP) scaffold at a second stage. For the experimental groups, a bioactive synthetic membrane embedded with BMP2, PDGF or both enveloped a PCL/ß-TCP scaffold was implanted in a single stage. Serial radiographs were taken at 1, 4, 8, and 12 weeks postoperatively from the definitive procedure and evaluated by two blinded observers using a previously described scoring system to judge union as primary outcome. RESULTS: All experimental groups demonstrated better union than the negative control (p = 0.01). The groups with BMP2 incorporated into the membrane demonstrated higher average union scores than the other groups (p = 0.01). The induced membrane group performed similarly to the PDGF group. Complete union was only demonstrated in groups with BMP2-eluting membranes. CONCLUSIONS: A synthetic membrane comprised of type 1 collagen embedded with controlled release BMP2 improved union of critical bone defects in a preclinical nonunion model.

Applying deep learning to quantify empty lacunae in histologic sections of osteonecrosis of the femoral head.

Osteonecrosis of the femoral head (ONFH) is a disease in which inadequate blood supply to the subchondral bone causes the death of cells in the bone marrow. Decalcified histology and assessment of the percentage of empty lacunae are used to quantify the severity of ONFH. However, the current clinical practice of manually counting cells is a tedious and inefficient process. We utilized the power of artificial intelligence by training an established deep convolutional neural network framework, Faster-RCNN, to automatically classify and quantify osteocytes (healthy and pyknotic) and empty lacunae in 135 histology images. The adjusted correlation coefficient between the trained cell classifier and the ground truth was R = 0.98. The methods detailed in this study significantly reduced the manual effort of cell counting in ONFH histological samples and can be translated to other fields of image quantification.

Probing the role of methyl methacrylate release from spacer materials in induced membrane bone healing.

In the induced membrane (IM) technique for bone reconstruction, a poly(methyl methacrylate) (PMMA) spacer is implanted to induce formation of a foreign body membrane around the defect site. Membrane development is essential for later bone grafting success, yet the mechanism by which the IM promotes bone regeneration remains unknown, as are the ways that spacer composition plays a role in the membrane's healing potential. This study investigated the impact of leached methyl methacrylate (MMA)-the major monomeric component of PMMA-on IM development. In vitro cell culture found that MMA elution did not impact endothelial cell or mesenchymal stem cell proliferation. For in vivo analysis, we advanced a streamlined rat femoral model to efficiently study the influence of spacer properties on IM characteristics. Comparison of membrane formation around polycaprolactone (PCL), MMA-eluting PCL (high-dose PCL-MMA and low-dose PCL-MMA), and surgical PMMA revealed robust membranes enveloped all groups after 4 weeks in vivo, with elevated expression of osteogenic bone morphogenetic protein-2 and angiogenic vascular endothelial growth factor compared with the surrounding muscle and bone tissues. Growth factor quantitation in IM tissue found no statistically significant difference between groups. New bone growth, vascularization, and CD163+ macrophage populations surrounding the polymer implants were also quantified; and blood vessel formation around high-dose PCL-MMA was found to be significantly decreased compared with PCL alone. To the best of our knowledge, these findings represent the first time that results have been obtained about the characteristics of membranes formed around PCL in the IM setting.

Effect of porosity of a functionally-graded scaffold for the treatment of corticosteroid-associated osteonecrosis of the femoral head in rabbits.

Background/Objective: Core decompression (CD) with scaffold and cell-based therapies is a promising strategy for providing both mechanical support and regeneration of the osteonecrotic area for early stage osteonecrosis of the femoral head (ONFH). We designed a new 3D printed porous functionally-graded scaffold (FGS) with a central channel to facilitate delivery of transplanted cells in a hydrogel to the osteonecrotic area. However, the optimal porous structural design for the FGS for the engineering of bone in ONFH has not been elucidated. The aim of this study was to fabricate and evaluate two different porous structures (30% or 60% porosity) of the FGSs in corticosteroid-associated ONFH in rabbits. METHODS: Two different FGSs with 30% or 60% porosity containing a 1-mm central channel were 3D printed using polycaprolactone and ß-tricalcium phosphate. The FGS was 3-mm diameter and 32-mm length and was composed of three segments: 1-mm in length for the non-porous proximal segment, 22-mm in length for the porous (30% versus 60%) middle segment, and 9-mm in length for the 15% porous distal segment. Eighteen male New Zealand White rabbits were given a single dose of 20 â€‹mg/kg methylprednisolone acetate intramuscularly. Four weeks later, rabbits were divided into three groups: the CD group, the 30% porosity FGS group, and the 60% porosity FGS group. In the CD group, a 3-mm diameter drill hole was created into the left femoral head. In the FGS groups, a 30% or 60% porosity implant was inserted into the bone tunnel. Eight weeks postoperatively, femurs were harvested and microCT, mechanical, and histological analyses were performed. RESULTS: The actual porosity and pore size of the middle segments were 26.4% â€‹± â€‹2.3% and 699 â€‹± â€‹56 â€‹µm in the 30% porosity FGS, and 56.0% â€‹± â€‹4.5% and 999 â€‹± â€‹71 â€‹µm in the 60% porosity FGS, respectively using microCT analysis. Bone ingrowth ratio in the 30% porosity FGS group was 73.9% â€‹± â€‹15.8%, which was significantly higher than 39.5% â€‹± â€‹13.0% in the CD group on microCT (p â€‹< â€‹0.05). Bone ingrowth ratio in the 60% porosity FGS group (61.3% â€‹± â€‹30.1%) showed no significant differences compared to the other two groups. The stiffness at the bone tunnel site in the 30% porosity FGS group was 582.4 â€‹± â€‹192.3 â€‹N/mm3, which was significantly higher than 338.7 â€‹± â€‹164.6 â€‹N/mm3 in the 60% porosity FGS group during push-out testing (p â€‹< â€‹0.05). Hematoxylin and eosin staining exhibited thick and mature trabecular bone around the porous FGS in the 30% porosity FGS group, whereas thinner, more immature trabecular bone was seen around the porous FGS in the 60% porosity FGS group. CONCLUSION: These findings indicate that the 30% porosity FGS may enhance bone regeneration and have superior biomechanical properties in the bone tunnel after CD in ONFH, compared to the 60% porosity FGS. TRANSLATION POTENTIAL STATEMENT: The translational potential of this article: This FGS implant holds promise for improving outcomes of CD for early stage ONFH.

Combining a Vascular Bundle and 3D Printed Scaffold with BMP-2 Improves Bone Repair and Angiogenesis.

Vascularization is currently considered the biggest challenge in bone tissue engineering due to necrosis in the center of large scaffolds. We established a new expendable vascular bundle model to vascularize a three-dimensional printed channeled scaffold with and without bone morphogenetic protein-2 (BMP-2) for improved healing of large segmental bone defects. Bone formation and angiogenesis in an 8 mm critical-sized bone defect in the rat femur were significantly promoted by inserting a bundle consisting of the superficial epigastric artery and vein into the central channel of a large porous polycaprolactone scaffold. Vessels were observed sprouting from the vascular bundle inserted in the central tunnel. Although the regenerated bone volume in the group receiving the scaffold and vascular bundle was similar to that of the healthy femur, the rate of union of the group was not satisfactory (25% at 8 weeks). BMP-2 delivery was found to promote not only bone formation but also angiogenesis in the critical-sized bone defects. Both insertion of the vascular bundle alone and BMP-2 loading alone induced comparable levels of angiogenesis and when used in combination, significantly greater vascular volume was observed. These findings suggest a promising new modality of treatment in large bone defects. Level of Evidence: Therapeutic level I. Impact statement Vascularization is currently the main challenge in bone tissue engineering. The combination of a vascular bundle and an osteoinductive three-dimensional printed graft significantly improved and accelerated bone regeneration and angiogenesis in critical-sized large bone defects, suggesting a promising new modality of treatment in large bone defects.

The efficacy of core decompression for steroid-associated osteonecrosis of the femoral head in rabbits.

Although core decompression (CD) is often performed in the early stage of osteonecrosis of the femoral head (ONFH), the procedure does not always prevent subsequent deterioration and the effects of CD are not fully clarified. The aim of this study is to evaluate the efficacy of CD for steroid-associated ONFH in rabbits. Twelve male and 12 female New Zealand rabbits were injected intramuscularly 20 mg/kg of methylprednisolone once and were divided into the disease control and CD groups. In the disease control group, rabbits had no treatment and were euthanized at 12 weeks postinjection. In the CD group, rabbits underwent left femoral CD at 4 weeks postinjection and were euthanized 8 weeks postoperatively. The left femurs were collected to perform morphological, biomechanical, and histological analysis. Bone mineral density and bone volume fraction in the femoral head in the CD group were significantly higher than in the disease control group. However, no difference in the mechanical strength was observed between the two groups. Histological analysis showed that alkaline phosphatase and CD31 positive cells significantly increased in the males after CD treatment. The number of empty lacunae in the surrounding trabecular bone was significantly higher in the CD group. The current study indicated that CD improved the morphological properties, but did not improve the mechanical strength in the femoral head at early-stage ONFH. These data suggest the need for additional biological, mechanical strategies, and therapeutic windows to improve the outcome of early-stage steroid-associated ONFH.

Development of a Dual Hydrogel Model System for Vascularization.

Numerous hydrogel-based culture systems are used to create in vitro model for prevascularization. Hydrogels used to induce a microenvironment conducive to microvessel formation are typically soft and fast degradable, but often suffer from maintaining a lasting perfusable channel in vitro. Here, a dual hydrogel system that consists of photo-crosslinkable gelatin methacrylate (GelMA) and polyethylene glycol dimethacrylate (PEGDMA) is reported. GelMA hydrogels present soft and rapidly degradable properties and show microporous structures while PEGDMA is relatively stiff, almost nondegradable in vitro, and less porous. The dual hydrogel system is sequentially photo-crosslinked to construct an endothelial cell (EC)-lined perfusable PEGDMA channel and surrounding GelMA for endothelial vascular networks. Such dual hydrogel system exhibits seamless integration of the stiff PEGDMA channel and the surrounding soft GelMA, and facilitates rapid EC sprouting and extensive microvessel formation from a stable endothelium on the PEGDMA channel into the GelMA. Furthermore, diffusivity of biomolecules in the perfusable dual hydrogel system is affected by both the structural and physicochemical properties of the hydrogel system and the microvascular networks formed in the system. The establishment of the dual hydrogel system for vascularization holds great promise as an in vitro angiogenesis model and prevascularization strategy of large tissue constructs.

Vascularized Bone-Mimetic Hydrogel Constructs by 3D Bioprinting to Promote Osteogenesis and Angiogenesis.

Bone is a highly vascularized tissue with a unique and complex structure. Long bone consists of a peripheral cortical shell containing a network of channels for vascular penetration and an inner highly vascularized bone marrow space. Bioprinting is a powerful tool to enable rapid and precise spatial patterning of cells and biomaterials. Here we developed a two-step digital light processing technique to fabricate a bone-mimetic 3D hydrogel construct based on octacalcium phosphate (OCP), spheroids of human umbilical vein endothelial cells (HUVEC), and gelatin methacrylate (GelMA) hydrogels. The bone-mimetic 3D hydrogel construct was designed to consist of a peripheral OCP-containing GelMA ring to mimic the cortical shell, and a central GelMA ring containing HUVEC spheroids to mimic the bone marrow space. We further demonstrate that OCP, which is evenly embedded in the GelMA, stimulates the osteoblastic differentiation of mesenchymal stem cells. We refined the design of a spheroid culture device to facilitate the rapid formation of a large number of HUVEC spheroids, which were embedded into different concentrations of GelMA hydrogels. It is shown that the concentration of GelMA modulates the extent of formation of the capillary-like structures originating from the HUVEC spheroids. This cell-loaded hydrogel-based bone construct with a biomimetic dual ring structure can be potentially used for bone tissue engineering.

Preclinical induced membrane model to evaluate synthetic implants for healing critical bone defects without autograft.

Critical bone defects pose a formidable orthopaedic problem in patients with bone loss. We developed a preclinical model based on the induced membrane technique using a synthetic graft to replace autograft for healing critical bone defects. Additionally, we used a novel osteoconductive scaffold coupled with a synthetic membrane to evaluate the potential for single-stage bone regeneration. Three experimental conditions were investigated in critical femoral defects in rats. Group A underwent a two-stage procedure with insertion of a polymethylmethacrylate (PMMA) spacer followed by replacement with a 3D printed polycaprolactone(PCL)/ß-tricalcium phosphate (ß-TCP) osteoconductive scaffold after 4 weeks. Group B received a single-stage PCL/ß-TCP scaffold wrapped in a PCL-based microporous polymer film creating a synthetic membrane. Group C received a single-stage bare PCL/ß-TCP scaffold. All groups were examined by serial radiographs for callus formation. After 12 weeks, the femurs were explanted and analyzed with micro-CT and histology. Mean callus scores tended to be higher in Group A. Group A showed statistically significant greater bone formation on micro-CT compared with other groups, although bone volume fraction was similar between groups. Histology results suggested extensive bone ingrowth and new bone formation within the macroporous scaffolds in all groups and cell infiltration into the microporous synthetic membrane. This study supports the use of a critical size femoral defect in rats as a suitable model for investigating modifications to the induced membrane technique without autograft harvest. Future investigations should focus on bioactive synthetic membranes coupled with growth factors for single-stage bone healing. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res.

The effects of a functionally-graded scaffold and bone marrow-derived mononuclear cells on steroid-induced femoral head osteonecrosis.

Osteonecrosis of the femoral head (ONFH) is a debilitating disease that may progress to femoral head collapse and subsequently, degenerative arthritis. Although injection of bone marrow-derived mononuclear cells (BMMCs) is often performed with core decompression (CD) in the early stage of ONFH, these treatments are not always effective in prevention of disease progression and femoral head collapse. We previously described a novel 3D printed, customized functionally-graded scaffold (FGS) that improved bone growth in the femoral head after CD in a normal healthy rabbit, by providing structural and mechanical guidance. The present study demonstrates similar results of the FGS in a rabbit steroid-induced osteonecrosis model. Furthermore, the injection of BMMCs into the CD decreased the osteonecrotic area in the femoral head. Thus, the combination of FGS and BMMC provides a new therapy modality that may improve the outcome of CD for early stage of ONFH by providing both enhanced biological and biomechanical cues to promote bone regeneration in the osteonecrotic area.

Three-dimensional fabrication of cell-laden biodegradable poly(ethylene glycol-co-depsipeptide) hydrogels by visible light stereolithography.

Stereolithography (SLA) holds great promise in fabrication of cell-laden hydrogels with biomimetic complexity for use in tissue engineering and pharmaceutics. However, the availability of biodegradable photocrosslinkable hydrogel polymers for SLA is very limited. In this study, a water-soluble methacrylated poly(ethylene glycol-co-depsipeptide) was synthesized to yield a biodegradable photocrosslinkable macromer for SLA. Structural analysis confirmed the inclusion of biodegradable peptide and ester groups and photocrosslinkable methacrylate groups into the polymer backbone. The new macromer combined with RGDS peptide was used for SLA fabrication of hydrogels in absence and presence of cells. With the increasing light exposure time in SLA, mechanical stiffness of the hydrogels increased from 3 ± 1 kPa to 38 ± 13 kPa. Total mass loss of the samples within 7 days in PBS was 13%-21% and within 24 days 35%-66%. Due to degradation, the mechanical stiffness decreased by one order magnitude within 7-day incubation in PBS. Encapsulated endothelial cells proliferated in the hydrogels during 10-day in vitro cell culturing study. The macromer was further used in SLA to fabricate bifurcating tubular structures as preliminary vessel grafts. The new biodegradable, photocrosslinkable polymer is a significant addition to the very limited material selection currently available for SLA-based fabrication of cell-laden tissue engineering constructs.

Optofluidic detection for cellular phenotyping.

Quantitative analysis of the output of processes and molecular interactions within a single cell is highly critical to the advancement of accurate disease screening and personalized medicine. Optical detection is one of the most broadly adapted measurement methods in biological and clinical assays and serves cellular phenotyping. Recently, microfluidics has obtained increasing attention due to several advantages, such as small sample and reagent volumes, very high throughput, and accurate flow control in the spatial and temporal domains. Optofluidics, which is the attempt to integrate optics with microfluidics, shows great promise to enable on-chip phenotypic measurements with high precision, sensitivity, specificity, and simplicity. This paper reviews the most recent developments of optofluidic technologies for cellular phenotyping optical detection.

Breast cancer cells induce cancer-associated fibroblasts to secrete hepatocyte growth factor to enhance breast tumorigenesis.

It has been well documented that microenvironment consisting of stroma affects breast cancer progression. However, the mechanisms by which cancer cells and fibroblasts, the major cell type in stroma, interact with each other during tumor development remains to be elucidated. Here, we show that the human cancer-associated fibroblasts (CAFs) had higher activity in enhancing breast tumorigenecity compared to the normal tissue-associated fibroblasts (NAFs) isolated from the same patients. The expression level of hepatocyte growth factor (HGF) in these fibroblasts was positively correlated with their ability to enhance breast tumorigenesis in mice. Deprivation of HGF using a neutralizing antibody reduced CAF-mediated colony formation of human breast cancer cells, indicating that CAFs enhanced cancer cell colony formation mainly through HGF secretion. Co-culture with human breast cancer MDA-MB-468 cells in a transwell system enhanced NAFs to secret HGF as well as promote tumorigenecity. The newly gained ability of these "educated" NAFs became irreversible after continuing this process till fourth passage. These results suggested that breast cancer cells could alter the nature of its surrounding fibroblasts to secrete HGF to support its own progression through paracrine signaling.

Multiple lineages of human breast cancer stem/progenitor cells identified by profiling with stem cell markers.

Heterogeneity of cancer stem/progenitor cells that give rise to different forms of cancer has been well demonstrated for leukemia. However, this fundamental concept has yet to be established for solid tumors including breast cancer. In this communication, we analyzed solid tumor cancer stem cell markers in human breast cancer cell lines and primary specimens using flow cytometry. The stem/progenitor cell properties of different marker expressing-cell populations were further assessed by in vitro soft agar colony formation assay and the ability to form tumors in NOD/SCID mice. We found that the expression of stem cell markers varied greatly among breast cancer cell lines. In MDA-MB-231 cells, PROCR and ESA, instead of the widely used breast cancer stem cell markers CD44(+)/CD24(-/low) and ALDH, could be used to highly enrich cancer stem/progenitor cell populations which exhibited the ability to self renew and divide asymmetrically. Furthermore, the PROCR(+)/ESA(+) cells expressed epithelial-mesenchymal transition markers. PROCR could also be used to enrich cells with colony forming ability from MB-361 cells. Moreover, consistent with the marker profiling using cell lines, the expression of stem cell markers differed greatly among primary tumors. There was an association between metastasis status and a high prevalence of certain markers including CD44(+)/CD24(-/low), ESA(+), CD133(+), CXCR4(+) and PROCR(+) in primary tumor cells. Taken together, these results suggest that similar to leukemia, several stem/progenitor cell-like subpopulations can exist in breast cancer.