Characterization of murine subacromial bursal-derived cells.

PURPOSE/AIM: The purpose of this study is to identify a cell population within the murine subcromial bursal-derived cells with characteristics compatible to an accepted mesenchymal stem cell description given by the International Society for Cellular Therapy (ISCT). MATERIALS AND METHODS: Murine subacromial bursa was harvested using microsurgical technique. Subacromial bursal-derived cells were classified through colony-forming units, microscopic morphology, fluorescent-activated cell sorting, and differentiation into chondrogenic, adipogenic, and osteogenic lineages. RESULTS: Subacromial bursal samples exhibited cell growth out of the tissue for an average of 115 ± 29 colony-forming units per 1 mL of complete media. Subacromial bursal-derived cells exhibited a long, spindle-shaped, fibroblast-like morphology. Subacromial bursal-derived cells positively expressed mesenchymal stem cell markers CD73, CD90, and CD105, and negatively expressed mesenchymal stem cell markers CD31 and CD45. Subacromial bursal-derived cells, examined by Image J analysis and quantitative gene expression, were found to differentiate into chondrogenic, adipogenic, and osteogenic lineages. CONCLUSIONS: This study demonstrated the feasibility of harvesting murine subacromial bursal tissue and identified a cell population within the subacromial bursa with characteristics compatible to an accepted mesenchymal stem cell description. The results of this study suggest that the mouse subacromial bursal-derived cell population harbors mesenchymal stem cells. Murine subacromial bursal tissue is a potential source for obtaining cells with mesenchymal stem cell characteristics for future utilization in orthopedic research to look into treatment of rotator cuff pathology.

The glucagon-like peptide 1 receptor agonist Exendin-4 induces tenogenesis in human mesenchymal stem cells.

Tendon injuries are common and account for up to 50% of musculoskeletal injuries in the United States. The poor healing nature of the tendon is attributed to poor vascularization and cellular composition. In the absence of FDA-approved growth factors for tendon repair, engineering strategies using bioactive factors, donor cells, and delivery matrices to promote tendon repair and regeneration are being explored. Growth factor alternatives in the form of small molecules, donor cells, and progenitors offer several advantages and enhance the tendon healing response. Small drug molecules and peptides offer stability over growth factors that are known to suffer from relatively short biological half-lives. The primary focus of this study was to assess the ability of the exendin-4 (Ex-4) peptide, a glucagon-like peptide 1 (GLP-1) receptor agonist, to induce tenocyte differentiation in bone marrow-derived human mesenchymal stem cells (hMSCs). We treated hMSCs with varied doses of Ex-4 in culture media to evaluate proliferation and tendonogenic differentiation. A 20 nM Ex-4 concentration was optimal for promoting cell proliferation and tendonogenic differentiation. Tendonogenic differentiation of hMSCs was evaluated via gene expression profile, immunofluorescence, and biochemical analyses. Collectively, the levels of tendon-related transcription factors (Mkx and Scx) and extracellular matrix (Col-I, Dcn, Bgn, and Tnc) genes and proteins were elevated compared to media without Ex-4 and other controls including insulin and IGF-1 treatments. The tendonogenic factor Ex-4 in conjunction with hMSCs appear to enhance tendon regeneration.

Subacromial bursa increases the failure force in a mouse model of supraspinatus detachment and repair.

BACKGROUND: It has been shown that subacromial bursa (SAB) harbors connective tissue progenitor cells. The purpose of this study was to evaluate the effects of implantation of SAB-derived cells (SBCs) suspended in a fibrin sealant bead and implantation of SAB tissue at rotator cuff repair site on biomechanical properties of the repair in a mouse (C57Bl/6) model of supraspinatus tendon (ST) detachment and repair. METHODS: Part 1: Murine SAB tissue was harvested and cultured. Viability of SBCs suspended in 10 µL of fibrin sealant beads was confirmed in vitro and in vivo. Eighty mice underwent right ST detachment and repair augmented with either fibrin sealant bead (control group) or fibrin sealant bead with 100,000 SBCs (study group) applied at the repair site. Part 2: 120 mice underwent right ST detachment and repair and were randomized equally into 4 groups: (1) a tissue group, which received a piece of freshly harvested SAB tissue; (2) a cell group, which received SBCs suspended in fibrin sealant bead; (3) a fibrin sealant group, which received plain fibrin sealant bead without cells; and (4) a control group, which received nothing at the ST repair site. An equal number of mice in each group were killed at 2 and 4 weeks. Specimens underwent biomechanical testing to evaluate failure force (part 1 and 2) and histologic analysis of the repair site (part 1 only). RESULTS: Part 1: The mean failure force in the study group was significantly higher than controls at 2 and 4 weeks (3.25 ± 1.03 N vs. 2.43 ± 0.56 N, P = .01, and 4.08 ± 0.99 N vs. 3.02 ± 0.8 N, P = .004, respectively). Mean cell density of the ST at the repair site was significantly lower in the study group at 2 weeks than in controls (18,292.13 ± 1706.41 vs. 29,501.90 ± 3627.49, P = .001). Study group specimens had lower proteoglycan contents than controls, but this difference was not statistically significant. Part 2: There was no difference in failure force between cell and tissue groups at the 2- and 4-week time points (P = .994 and P = .603, respectively). There was no difference in failure force between fibrin sealant bead and control groups at the 2- and 4-week time points (P = .978 and P = .752, respectively). CONCLUSION: This study shows that the application of SBCs and SAB tissue at the rotator cuff repair site increases the strength of repair in a murine model of rotator cuff detachment and repair.

Progress and challenges of graphene and its congeners for biomedical applications.

Nanomaterials by virtue of their small size and enhanced surface area, present unique physicochemical properties that enjoy widespread applications in bioengineering, biomedicine, biotechnology, disease diagnosis, and therapy. In recent years, graphene and its derivatives have attracted a great deal of attention in various applications, including photovoltaics, electronics, energy storage, catalysis, sensing, and biotechnology owing to their exceptional structural, optical, thermal, mechanical, and electrical. Graphene is a two-dimensional sheet of sp2 hybridized carbon atoms of atomic thickness, which are arranged in a honeycomb crystal lattice structure. Graphene derivatives are graphene oxide (GO) and reduced graphene oxide (rGO), which are highly oxidized and less oxidized forms of graphene, respectively. Another form of graphene is graphene quantum dots (GQDs), having a size of less than 20 nm. Contemporary graphene research focuses on using graphene nanomaterials for biomedical purposes as they have a large surface area for loading biomolecules and medicine and offer the potential for the conjugation of fluorescent dyes or quantum dots for bioimaging. The present review begins with the synthesis, purification, structure, and properties of graphene nanomaterials. Then, we focussed on the biomedical application of graphene nanomaterials with special emphasis on drug delivery, bioimaging, biosensing, tissue engineering, gene delivery, and chemotherapy. The implications of graphene nanomaterials on human health and the environment have also been summarized due to their exposure to their biomedical applications. This review is anticipated to offer useful existing understanding and inspire new concepts to advance secure and effective graphene nanomaterials-based biomedical devices.

Injectable RANKL sustained release formulations to accelerate orthodontic tooth movement.

BACKGROUND: Accelerating orthodontic tooth movement (OTM) through biologically effective methods, such as increasing osteoclast-mediated alveolar resorption, could effectively shorten treatment time. OBJECTIVE: To evaluate an injectable formulation containing receptor activator of nuclear factor kappa-B ligand (RANKL) on the OTM. MATERIALS AND METHODS: We fabricated a RANKL formulation from 100 µl of 100 µg/ml RANKL adsorbed on 10 mg of poly(lactic acid-co-glycolic acid) microspheres embedded in a 10 wt% aqueous hydroxyethyl cellulose carrier gel. We characterized these formulations for the rate of RANKL release, and then tested for bioactivity using in vitro cell culture. In vivo OTM studies were conducted using 15 week old male Wistar rats for 14 days. We injected the RANKL formulations palatal to the left maxillary first molar and accomplished OTM with a nickel-titanium (NiTi) coil spring applying 5-8 g force. Control groups involved the application of NiTi coil spring with and without placebo formulation. The outcome measure included the distance of tooth movement, bone volume fraction, tissue density, and root volume determined with micro-computed tomography. We determined the amount of osteoclast activity using tartrate-resistant acid phosphatase (TRAP) staining. RESULTS: These formulations were able to sustain the release of RANKL for more than 30 days, and the released RANKL showed a positive effect on mice osteoclast precursor cells (RAW 264.7). Reported injectable RANKL formulations were effective in accelerating OTM compared with other control groups, with 129.2 per cent more tooth movement than no formulation and 71.8 per cent more than placebo formulation, corresponding with a significant increase in the amount of TRAP activity. We did not observe any significant differences in root resorption between the groups. CONCLUSION: Our study shows a significant increase in OTM with injectable formulations containing RANKL.

Nanomaterials/Nanocomposites for Osteochondral Tissue.

For many years, the avascular nature of cartilage tissue has posed a clinical challenge for replacement, repair, and reconstruction of damaged cartilage within the human body. Injuries to cartilage and osteochondral tissues can be due to osteoarthritis, sports, aggressive cancers, and repetitive stresses and inflammation on wearing tissue. Due to its limited capacity for regeneration or repair, there is a need for suitable material systems which can recapitulate the function of the native osteochondral tissue physically, mechanically, histologically, and biologically. Tissue engineering (TE) approaches take advantage of principles of biomedical engineering, clinical medicine, and cell biology to formulate, functionalize, and apply biomaterial scaffolds to aid in the regeneration and repair of tissues. Nanomaterial science has introduced new methods for improving and fortifying TE scaffolds, and lies on the forefront of cutting-edge TE strategies. These nanomaterials enable unique properties directly correlated to their sub-micron dimensionality including structural and cellular advantages. Examples include electrospun nanofibers and emulsion nanoparticles which provide nanoscale features for biomaterials, more closely replicating the 3D extracellular matrix, providing better cell adhesion, integration, interaction, and signaling. This chapter aims to provide a detailed overview of osteochondral regeneration and repair using TE strategies with a focus on nanomaterials and nanocomposites.

Guided differentiation of bone marrow stromal cells on co-cultured cartilage and bone scaffolds.

Focal chondral defects that result from traumatic injuries to the knee remain one of the most common causes of disability in patients. Current solutions for healing focal cartilage defects are mainly limited by the production of inferior cartilage-like tissue and subsequent delamination due to incomplete healing of the subchondral bone. In this experiment a polymeric osteochondral implant for guiding autologous bone marrow stem cells (BMSCs) to populate the scaffold to create distinctive bone and cartilage tissue is used. The cartilage component presents bioactive aligned nanofibers containing chondroitin sulfate and hyaluronic acid while the bone component includes hydroxyapatite to promote chondrogenic and osteogenic differentiation of the rat BMSCs in vitro. The different cartilage and bone components resulted in the elevated expression of osteogenic markers such as bone sialoprotein, runt related transcription factor 2, and bone morphogenetic protein 2 in the deeper bone layer and chondrogenic markers such as collagen type II and aggrecan in the cartilage layer. Through immunofluorescence imaging, the alignment of the secreted collagen type II fibrils and aggrecan was visualized and quantified on the cartilage component of the scaffold. These current studies show that the biodegradable biphasic osteochondral implant may be effective in promoting more hyaline-like tissue to fill in chondral defects of the knee.

Properties of biologic scaffolds and their response to mesenchymal stem cells.

PURPOSE: The purpose of this study was to examine, in vitro, the cellular response of human mesenchymal stem cells (MSCs) to sample types of commercially available scaffolds in comparison with control, native tendon tissue (fresh-frozen rotator cuff tendon allograft). METHODS: MSCs were defined by (1) colony-forming potential; (2) ability to differentiate into tendon, cartilage, bone, and fat tissue; and (3) fluorescence-activated cell sorting analysis (CD73, CD90, CD45). Samples were taken from fresh-frozen human rotator cuff tendon (allograft), human highly cross-linked collagen membrane (Arthroflex; LifeNet Health, Virginia Beach, VA), porcine non-cross-linked collagen membrane (Mucograft; Geistlich Pharma, Lucerne, Switzerland), a human platelet-rich fibrin matrix (PRF-M), and a fibrin matrix based on platelet-rich plasma (ViscoGel; Arthrex, Naples, FL). Cells were counted for adhesion (24 hours), thymidine assay for cell proliferation (96 hours), and live/dead stain for viability (168 hours). Histologic analysis was performed after 21 days, and the unloaded scaffolds were scanned with electron microscopy. RESULTS: MSCs were successfully differentiated into all cell lines. A significantly greater number of cells adhered to both the non-cross-linked porcine collagen scaffold and PRF-M. Cell activity (proliferation) was significantly higher in the non-cross-linked porcine collagen scaffold compared with PRF-M and fibrin matrix based on platelet-rich plasma. There were no significant differences found in the results of the live/dead assay. CONCLUSIONS: Significant differences in the response of human MSCs to biologic scaffolds existed. MSC adhesion, proliferation, and scaffold morphology evaluated by histologic analysis and electron microscopy varied throughout the evaluated types of scaffolds. Non-cross-linked porcine collagen scaffolds showed superior results for cell adhesion and proliferation, as well as on histologic evaluation. CLINICAL RELEVANCE: This study enables the clinician and scientist to choose scaffold materials according to their specific interaction with MSCs.

Decellularized extracellular matrix biomaterials for regenerative therapies: Advances, challenges and clinical prospects.

Tissue engineering and regenerative medicine have shown potential in the repair and regeneration of tissues and organs via the use of engineered biomaterials and scaffolds. However, current constructs face limitations in replicating the intricate native microenvironment and achieving optimal regenerative capacity and functional recovery. To address these challenges, the utilization of decellularized tissues and cell-derived extracellular matrix (ECM) has emerged as a promising approach. These biocompatible and bioactive biomaterials can be engineered into porous scaffolds and grafts that mimic the structural and compositional aspects of the native tissue or organ microenvironment, both in vitro and in vivo. Bioactive dECM materials provide a unique tissue-specific microenvironment that can regulate and guide cellular processes, thereby enhancing regenerative therapies. In this review, we explore the emerging frontiers of decellularized tissue-derived and cell-derived biomaterials and bio-inks in the field of tissue engineering and regenerative medicine. We discuss the need for further improvements in decellularization methods and techniques to retain structural, biological, and physicochemical characteristics of the dECM products in a way to mimic native tissues and organs. This article underscores the potential of dECM biomaterials to stimulate in situ tissue repair through chemotactic effects for the development of growth factor and cell-free tissue engineering strategies. The article also identifies the challenges and opportunities in developing sterilization and preservation methods applicable for decellularized biomaterials and grafts and their translation into clinical products.

Stimuli-responsive peptide assemblies: Design, self-assembly, modulation, and biomedical applications.

Peptide molecules have design flexibility, self-assembly ability, high biocompatibility, good biodegradability, and easy functionalization, which promote their applications as versatile biomaterials for tissue engineering and biomedicine. In addition, the functionalization of self-assembled peptide nanomaterials with other additive components enhances their stimuli-responsive functions, promoting function-specific applications that induced by both internal and external stimulations. In this review, we demonstrate recent advance in the peptide molecular design, self-assembly, functional tailoring, and biomedical applications of peptide-based nanomaterials. The strategies on the design and synthesis of single, dual, and multiple stimuli-responsive peptide-based nanomaterials with various dimensions are analyzed, and the functional regulation of peptide nanomaterials with active components such as metal/metal oxide, DNA/RNA, polysaccharides, photosensitizers, 2D materials, and others are discussed. In addition, the designed peptide-based nanomaterials with temperature-, pH-, ion-, light-, enzyme-, and ROS-responsive abilities for drug delivery, bioimaging, cancer therapy, gene therapy, antibacterial, as well as wound healing and dressing applications are presented and discussed. This comprehensive review provides detailed methodologies and advanced techniques on the synthesis of peptide nanomaterials from molecular biology, materials science, and nanotechnology, which will guide and inspire the molecular level design of peptides with specific and multiple functions for function-specific applications.

Amorphous silica fiber matrix biomaterials: An analysis of material synthesis and characterization for tissue engineering.

Silica biomaterials including Bioglass offer great biocompatibility and bioactivity but fail to provide pore and degradation features needed for tissue engineering. Herein we report on the synthesis and characterization of novel amorphous silica fiber matrices to overcome these limitations. Amorphous silica fibers were fused by sintering to produce porous matrices. The effects of sacrificial polymer additives such as polyvinyl alcohol (PVA) and cellulose fibers (CF) on the sintering process were also studied. The resulting matrices formed between sintering temperatures of 1,350-1,550 °C retained their fiber structures. The matrices presented pores in the range of 50-200 µm while higher sintering temperatures resulted in increased pore diameter. PVA addition to silica significantly reduced the pore diameter and porosity compared with silica matrices with or without the addition of CF. The PVA additive morphologically appeared to fuse the silica fibers to a greater extent and resulted in significantly higher compressive modulus and strength than the rest of the matrices synthesized. These matrices lost roughly 30% of their original mass in an in vitro degradation study over 40 weeks. All matrices absorbed 500 wt% of water and did not change in their overall morphology, size, or shape with hydration. These fiber matrices supported human mesenchymal stem cell adhesion, proliferation, and mineralized matrix production. Amorphous silica fiber biomaterials/matrices reported here are biodegradable and porous and closely resemble the native extracellular matrix structure and water absorption capacity. Extending the methodology reported here to alter matrix properties may lead to a variety of tissue engineering, implant, and drug delivery applications.

NOVEL HIGH-STRENGTH POLYESTER COMPOSITE SCAFFOLDS FOR BONE REGENERATION.

Repair of critical sized bone defects, particularly in load-bearing areas, is a major clinical problem that requires surgical intervention and implantation of biological or engineered grafts. For load-bearing sites, it is essential to use engineered grafts that have both sufficient mechanical strength and appropriate pore properties to support bone repair and tissue regeneration. Unfortunately, the mechanical properties of such grafts are often compromised due to the creation of pores required to facilitate tissue ingrowth following implantation. To overcome the limitations associated with porous scaffolds and their reduced mechanical strength, we have developed a methodology for creating a solid structure that retains its bulk mechanical properties while also evolving into a porous structure in a biological environment through degradation and erosion. In this study, we utilized polyesters that have been approved by the FDA, including poly (lactic acid) (PLA), poly(glycolic acid) (PGA), their copolymer PLGA (PLGA, with a ratio of 85:15 and 50:50 of PLA:PGA), and poly(caprolactone) (PCL). These polymers and their ceramic composites with tricalcium phosphate (TCP) were compression molded into solid forms, which exhibited mechanical properties with compressive modulus as high as 2745 ± 364 MPa within the range of human trabecular bone and in the lower range of human cortical bone. The use of fast-degrading PLGA (50:50) and PGA as porogens allowed the formation of pores within the solid structures due to their degradation, and the TCP acts as a buffering agent to neutralize their acidic degradation byproducts. These scaffolds facilitated the growth of new blood vessels and tissue ingrowth in a subcutaneous implantation model. In addition, in a rat critical-sized mandibular bone defects these scaffolds supported bone growth with 70% of new bone volume fraction. Furthermore, the extent of bone regeneration was found to be higher for the scaffolds with bone morphogenic proteins (BMP2), indicating their suitability for bone repair and regeneration.

Novel Injectable Fluorescent Polymeric Nanocarriers for Intervertebral Disc Application.

Damage to intervertebral discs (IVD) can lead to chronic pain and disability, and no current treatments can fully restore their function. Some non-surgical treatments have shown promise; however, these approaches are generally limited by burst release and poor localization of diverse molecules. In this proof-of-concept study, we developed a nanoparticle (NP) delivery system to efficiently deliver high- and low-solubility drug molecules. Nanoparticles of cellulose acetate and polycaprolactone-polyethylene glycol conjugated with 1-oxo-1H-pyrido [2,1-b][1,3]benzoxazole-3-carboxylic acid (PBC), a novel fluorescent dye, were prepared by the oil-in-water emulsion. Two drugs, a water insoluble indomethacin (IND) and a water soluble 4-aminopyridine (4-AP), were used to study their release patterns. Electron microscopy confirmed the spherical nature and rough surface of nanoparticles. The particle size analysis revealed a hydrodynamic radius ranging ~150-162 nm based on dynamic light scattering. Zeta potential increased with PBC conjugation implying their enhanced stability. IND encapsulation efficiency was almost 3-fold higher than 4-AP, with release lasting up to 4 days, signifying enhanced solubility, while the release of 4-AP continued for up to 7 days. Nanoparticles and their drug formulations did not show any apparent cytotoxicity and were taken up by human IVD nucleus pulposus cells. When injected into coccygeal mouse IVDs in vivo, the nanoparticles remained within the nucleus pulposus cells and the injection site of the nucleus pulposus and annulus fibrosus of the IVD. These fluorescent nano-formulations may serve as a platform technology to deliver therapeutic agents to IVDs and other tissues that require localized drug injections.

Smart Orthopedic Biomaterials and Implants.

Musculoskeletal injuries including bone defects continue to present a significant challenge in orthopedic surgery due to suboptimal healing. Bone reconstruction strategies focused on the use of biological grafts and bone graft substitutes in the form of biomaterials-based 3D structures in fracture repair. Recent advances in biomaterials science and engineering have resulted in the creation of intricate 3D bone-mimicking structures that are mechanically stable, biodegradable, and bioactive to support bone regeneration. Current efforts are focused on improving the biomaterial and implant physicochemical properties to promote interactions with the host tissue and osteogenesis. The "smart" biomaterials and their 3D structures are designed to actively interact with stem/progenitor cells and the extracellular matrix (ECM) to influence the local environment towards osteogenesis and de novo tissue formation. This article will summarize such smart biomaterials and the methodologies to apply either internal or external stimuli to control the tissue healing microenvironment. A particular emphasis is also made on the use of smart biomaterials and strategies to create functional bioactive implants for bone defect repair and regeneration.

Nanofiber matrix formulations for the delivery of Exendin-4 for tendon regeneration: In vitro and in vivo assessment.

Tendon and ligament injuries are the most common musculoskeletal injuries, which not only impact the quality of life but result in a massive economic burden. Surgical interventions for tendon/ligament injuries utilize biological and/or engineered grafts to reconstruct damaged tissue, but these have limitations. Engineered matrices confer superior physicochemical properties over biological grafts but lack desirable bioactivity to promote tissue healing. While incorporating drugs can enhance bioactivity, large matrix surface areas and hydrophobicity can lead to uncontrolled burst release and/or incomplete release due to binding. To overcome these limitations, we evaluated the delivery of a peptide growth factor (exendin-4; Ex-4) using an enhanced nanofiber matrix in a tendon injury model. To overcome drug surface binding due to matrix hydrophobicity of poly(caprolactone) (PCL)-which would be expected to enhance cell-material interactions-we blended PCL and cellulose acetate (CA) and electrospun nanofiber matrices with fiber diameters ranging from 600 to 1000 nm. To avoid burst release and protect the drug, we encapsulated Ex-4 in the open lumen of halloysite nanotubes (HNTs), sealed the HNT tube endings with a polymer blend, and mixed Ex-4-loaded HNTs into the polymer mixture before electrospinning. This reduced burst release from ∼75% to ∼40%, but did not alter matrix morphology, fiber diameter, or tensile properties. We evaluated the bioactivity of the Ex-4 nanofiber formulation by culturing human mesenchymal stem cells (hMSCs) on matrix surfaces for 21 days and measuring tenogenic differentiation, compared with nanofiber matrices in basal media alone. Strikingly, we observed that Ex-4 nanofiber matrices accelerated the hMSC proliferation rate and elevated levels of sulfated glycosaminoglycan, tendon-related genes (Scx, Mkx, and Tnmd), and ECM-related genes (Col-I, Col-III, and Dcn), compared to control. We then assessed the safety and efficacy of Ex-4 nanofiber matrices in a full-thickness rat Achilles tendon defect with histology, marker expression, functional walking track analysis, and mechanical testing. Our analysis confirmed that Ex-4 nanofiber matrices enhanced tendon healing and reduced fibrocartilage formation versus nanofiber matrices alone. These findings implicate Ex-4 as a potentially valuable tool for tendon tissue engineering.

Fluorescent liposomal nanocarriers for targeted drug delivery in ischemic stroke therapy.

Ischemic stroke causes acute CNS injury and long-term disability, with limited treatment options such as surgical clot removal or clot-busting drugs. Neuroprotective therapies are needed to protect vulnerable brain regions. The purinergic receptor P2X4 is activated during stroke and exacerbates post-stroke damage. The chemical compound 5-(3-Bromophenyl)-1,3-dihydro-2H-Benzofuro[3,2-e]-1,4-diazepin-2-one (5BDBD) inhibits P2X4 and has shown neuroprotective effects in rodents. However, it is difficult to formulate for systemic delivery to the CNS. The current manuscript reports for the first time, the synthesis and characterization of 5BDBD PEGylated liposomal formulations and evaluates their feasibility to treat stroke in a preclinical mice model. A PEGylated liposomal formulation of 5BDBD was synthesized and characterized, with encapsulation efficacy of >80%, and release over 48 hours. In vitro and in vivo experiments with Nile red encapsulation showed cytocompatibility and CNS infiltration of nanocarriers. Administered 4 or 28 hours after stroke onset, the nanoformulation provided significant neuroprotection, reducing infarct volume by ∼50% compared to controls. It outperformed orally-administered 5BDBD with a lower dose and shorter treatment duration, suggesting precise delivery by nanoformulation improves outcomes. The fluorescent nanoformulations may serve as a platform for delivering and tracking therapeutic agents for stroke treatment.

FKBP38 Regulates Self-Renewal and Survival of GBM Neurospheres.

Glioblastoma is the most common malignant primary brain tumor. The outcome is dismal, despite the multimodal therapeutic approach that includes surgical resection, followed by radiation and chemotherapy. The quest for novel therapeutic targets to treat glioblastoma is underway. FKBP38, a member of the immunophilin family of proteins, is a multidomain protein that plays an important role in the regulation of cellular functions, including apoptosis and autophagy. In this study, we tested the role of FKBP38 in glioblastoma tumor biology. Expression of FKBP38 was upregulated in the patient-derived primary glioblastoma neurospheres (GBMNS), compared to normal human astrocytes. Attenuation of FKBP38 expression decreased the viability of GBMNSs and increased the caspase 3/7 activity, indicating that FKBP38 is required for the survival of GBMNSs. Further, the depletion of FKBP38 significantly reduced the number of neurospheres that were formed, implying that FKBP38 regulates the self-renewal of GBMNSs. Additionally, the transient knockdown of FKBP38 increased the LC3-II/I ratio, suggesting the induction of autophagy with the depletion of FKBP38. Further investigation showed that the negative regulation of autophagy by FKBP38 in GBMNSs is mediated through the JNK/C-Jun-PTEN-AKT pathway. In vivo, FKBP38 depletion significantly extended the survival of tumor-bearing mice. Overall, our results suggest that targeting FKBP38 imparts an anti-glioblastoma effect by inducing apoptosis and autophagy and thus can be a potential therapeutic target for glioblastoma therapy.

3D bioprinting and photocrosslinking: emerging strategies & future perspectives.

3D bioprinting has enabled the creation of biomimetic tissue constructs for regenerative medicine and in vitro model systems. Large-scale production of 3D structures at the micron-scale resolution is achieved through bioprinting using custom bioinks. Stability and 3D construct compliance play an important role in offering cells with biomechanical cues that regulate their behavior and enable in vivo implantation. Various crosslinking strategies are developed to stabilize the 3D printed structures and new methodologies are constantly being evaluated to overcome the limitations of the existing methods. Photo-crosslinking has emerged as a simple and elegant technique that offers precise control over the spatiotemporal gelation of bioinks during bioprinting. This article summarizes the use of photo-crosslinking agents and methodology towards optimizing 3D constructs for specific biomedical applications. The article also takes into account various bioinks and photo-crosslinkers in creating stable 3D printed structures that offer bioactivity with desirable physicochemical properties. The current challenges of 3D bioprinting and new directions that can advance the field in its wide applicability to create 3D tissue models to study diseases and organ transplantation are also summarized.

Insulin-Functionalized Bioactive Fiber Matrices with Bone Marrow-Derived Stem Cells in Rat Achilles Tendon Regeneration.

Approximately half of annual musculoskeletal injuries in the US involve tendon tears. The naturally hypocellular and hypovascular tendon environment makes tendons injury-prone and heal slowly. Tendon tissue engineering strategies often use biomimetic scaffolds combined with bioactive factors and/or cells to enhance healing. FDA-approved growth factors to promote tendon healing are lacking, which highlights the need for safe and effective bioactive factors. Our previous work evaluated insulin as a bioactive factor and identified an optimal dose to promote in vitro mesenchymal stem cell survival, division, and tenogenesis. The present work evaluates the ability of insulin-functionalized electrospun nanofiber matrices with or without mesenchymal stem cells to enhance tendon repair in a rat Achilles injury model. Electrospun nanofiber matrices were functionalized with insulin, cultured with or without mesenchymal stem cells, and sutured to transected Achilles tendons in rats. We analyzed rat tendons 4 and 8 weeks after surgery for the tendon morphology, collagen production, and mechanical properties. Bioactive insulin-functionalized fiber matrices with mesenchymal stem cells resulted in significantly increased collagen I and III at 4 and 8 weeks postsurgery. Additionally, these matrices supported highly aligned collagen fibrils in the regenerated tendon tissue at 8 weeks. However, treatment- and control-regenerated tissues had similar tensile properties at 8 weeks, which were less than that of the native Achilles tendon. Our preliminary results establish the benefits of insulin-functionalized fiber matrices in promoting higher levels of collagen synthesis and alignment needed for functional recovery of tendon repair.

Natural Polymer-Based Micronanostructured Scaffolds for Bone Tissue Engineering.

Although bone tissue allografts and autografts aremoften used as a regenerative tissue during the bone healing, their availability, donor site morbidity, and immune response to grafted tissue are limiting factors their more common usage. Tissue engineered implants, such as acellular or cellular polymeric structures, can be an alternative solution. A variety of scaffold fabrication techniques including electrospinning, particulate leaching, particle sintering, and more recently 3D printing have been used to create scaffolds with interconnected pores and mechanical properties for tissue regeneration. Simply combining particle sintering and molecular self-assembly to create porous microstructures with imbued nanofibers to produce micronanostructures for tissue regeneration applications. Natural polymers like polysaccharides, proteins and peptides of plant or animal origin have gained significant attention due to their assured biocompatibility in tissue regeneration. However, majority of these polymers are water soluble and structures derived from them are in the form of hydrogels and require additional stabilization via cross-linking. For bone healing applications scaffolds are required to be strong, and support attachment, proliferation and differentiation of osteoprogenitors into osteoblasts. Our ongoing work utilizes plant polysaccharide cellulose derivatives and collagen to create mechanically stable and bioactive micronanostructured scaffold for bone tissue engineering. Scaffold microstructure is essentially solvent sintered cellulose acetate (CA) microspheres in the form of a negative template for trabecular bone with defined pore and mechanical properties. Collagen nanostructures are imbued into the 3D environment of CA scaffolds using collagen molecular self-assembly principles. The resultant CA-collagen micronanostructures provide the benefits of combined polymers and serve as an alternative material platform to many FDA approved polyesters. Our ongoing studies and published work confirm improved osteoprogenitor adhesion, proliferation, migration, differentiation, extracellular matrix (ECM) secretion in promoting bone healing. In this chapter we will provide a detailed protocol on the creation of micronanostructured CA-collagen scaffolds and their characterization for bone tissue engineering using human mesenchymal stem cells.