Xenogenic Implantation of Human Mesenchymal Stromal Cells Using a Novel 3D-Printed Scaffold of PLGA and Graphene Leads to a Significant Increase in Bone Mineralization in a Rat Segmental Femoral Bone Defect.

Tissue-engineering technologies have the potential to provide an effective approach to bone regeneration. Based on the published literature and data from our laboratory, two biomaterial inks containing PLGA and blended with graphene nanoparticles were fabricated. The biomaterial inks consisted of two forms of commercially available PLGA with varying ratios of LA:GA (65:35 and 75:25) and molecular weights of 30,000-107,000. Each of these forms of PLGA was blended with a form containing a 50:50 ratio of LA:GA, resulting in ratios of 50:65 and 50:75, which were subsequently mixed with a 0.05 wt% low-oxygen-functionalized derivative of graphene. Scanning electron microscopy showed interconnected pores in the lattice structures of each scaffold. The cytocompatibility of human ADMSCs transduced with a red fluorescent protein (RFP) was evaluated in vitro. The in vivo biocompatibility and the potential to repair bones were evaluated in a critically sized 5 mm mechanical load-bearing segmental femur defect model in rats. Bone repair was monitored by radiological, histological, and microcomputed tomography methods. The results showed that all of the constructs were biocompatible and did not exhibit any adverse effects. The constructs containing PLGA (50:75)/graphene alone and with hADMSCs demonstrated a significant increase in mineralized tissues within 60 days post-treatment. The percentage of bone volume to total volume from microCT analyses in the rats treated with the PLGA + cells construct showed a 50% new tissue formation, which matched that of a phantom. The microCT results were supported by Von Kossa staining.

Mesenchymal Stem Cell Use in Acute Tendon Injury: In Vitro Tenogenic Potential vs. In Vivo Dose Response.

Stem cell therapy for the treatment of tendon injury is an emerging clinical practice in the fields of human and veterinary sports medicine; however, the therapeutic benefit of intralesional transplantation of mesenchymal stem cells in tendonitis cases is not well designed. Questions persist regarding the overall tenogenic potential and efficacy of this treatment alone. In this study, we aimed to isolate a rat mesenchymal stem cell lineage for in vitro and in vivo use, to assess the effects of growth factor exposure in vitro on cell morphology, behavior, and tendon-associated glycoprotein production, and to assess the therapeutic potential of intralesional stem cells, as a function of dose, in vivo. First, rat adipose-derived (rAdMSC) and bone marrow-derived (rBMSC) stem cell lineages were isolated, characterized with flow cytometric analysis, and compared in terms of proliferation (MTS assay) and cellular viability (calcein AM staining). Rat AdMSCs displayed superior proliferation and more homogenous CD 73, CD 44H, and CD 90 expression as compared to rBMSC. Next, the tenogenic differentiation potential of the rAdMSC lineage was tested in vitro through isolated and combined stimulation with reported tenogenic growth factors, transforming growth factor (TGF)-ß3 and connective tissue growth factor (CTGF). We found that the most effective tenogenic factor in terms of cellular morphologic change, cell alignment/orientation, sustained cellular viability, and tendon-associated glycoprotein upregulation was TGFß3, and we confirmed that rAdMSC could be induced toward a tenogenic lineage in vitro. Finally, the therapeutic potential of rAdMSCs as a function of dose was assessed using a rat acute Achilles tendon injury model. Amounts of 5 × 105 (low dose) and 4 × 106 (high dose) were used. Subjectively, on the gross morphology, the rAdMSC-treated tendons exhibited fewer adhesions and less scar tissue than the control tendons; however, regardless of the rAdMSC dose, no significant differences in histological grade or tissue collagen I deposition were noted between the rAdMSC-treated and control tendons. Collectively, rAdMSCs exhibited appropriate stem cell markers and tenogenic potential in vitro, but the clinical efficacy of intralesional implantation of undifferentiated cells in acute tendonitis cases could not be proven. Further investigation into complementary therapeutics or specialized culture conditions prior to implantation are warranted.

Effects on Tissue Integration of Collagen Scaffolds Used for Local Delivery of Gentamicin in a Rat Mandible Defect Model.

Surgical site infections (SSIs) are a common complication following orthopedic surgery. SSIs may occur secondary to traumatic or contaminated wounds or may result from invasive procedures. The development of biofilms is often associated with implanted materials used to stabilize injuries and to facilitate healing. Regardless of the source, SSIs can be challenging to treat. This has led to the development of devices that act simultaneously as local antibiotic delivery vehicles and as scaffolds for tissue regeneration. The goal for the aforementioned devices is to increase local drug concentration in order to enhance bactericidal activity while reducing the risk of systemic side effects and toxicity from the administered drug. The aims of this study were to assess the effect of antibiotic loading of a collagen matrix on the tissue integration of the matrix using a rat mandibular defect model. We hypothesized that the collagen matrix could load and elute gentamicin, that the collagen matrix would be cytocompatible in vitro, and that the local delivery of a high dose of gentamicin via loaded collagen matrix would negatively impact the tissue-scaffold interface. The results indicate that the collagen matrix could load and elute the antimicrobial gentamicin and that it was cytocompatible in vitro with or without the presence of gentamicin and found no significant impact on the tissue-scaffold interface when the device was loaded with a high dose of gentamicin.

In vitro and in vivo assessment of caprine origin Staphylococcus aureus ST398 strain UTCVM1 as an osteomyelitis pathogen.

Staphylococcus aureus (SA) is a significant and well-recognized causative organism of bacterial osteomyelitis. Osteomyelitis is an inflammatory bone disease characterized by progressive bone destruction and loss. This disease causes significant morbidity and mortality to the patient and poses therapeutic challenges for clinicians. To improve the efficacy of therapeutic strategies to combat bacterial osteomyelitis, there is a need to define the molecular epidemiology of bacterial organisms more clearly and further the understanding of the pathogenesis of SA osteomyelitis. We conducted in vitro characterization of the pathogenic capabilities of an isolate of SA ST398 derived from a clinical case of osteomyelitis in a goat. We also report a rodent mandibular defect model to determine the ability of ST398 to cause reproducible osteomyelitis. Our results indicate that ST398 can invade and distort pre-osteoblastic cells in culture, induce significant inflammation and alter expression of osteoregulatory cytokines. We also demonstrate the ability of ST398 to induce osteomyelitis in a rat mandibular model. When compiled, these data support ST398 as a competent osteomyelitis pathogen.

Corrigendum: In vitro and In vivo assessment of caprine origin Staphylococcus aureus ST398 strain UTCVM1 as an osteomyelitis pathogen.

[This corrects the article DOI: 10.3389/fcimb.2022.1015655.].

Temporal metabolic profiling of bone healing in a caprine tibia segmental defect model.

Bone tissue engineering is an emerging field of regenerative medicine, with a wide array of biomaterial technologies and therapeutics employed. However, it is difficult to objectively compare these various treatments during various stages of tissue response. Metabolomics is rapidly emerging as a powerful analytical tool to establish broad-spectrum metabolic signatures for a target biological system. Developing an effective biomarker panel for bone repair from small molecule data would provide an objective metric to readily assess the efficacy of novel therapeutics in relation to natural healing mechanisms. In this study we utilized a large segmental bone defect in goats to reflect trauma resulting in substantial volumetric bone loss. Characterization of the native repair capacity was then conducted over a period of 12 months through the combination of standard (radiography, computed tomography, histology, biomechanics) data and ultra-high-performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) metabolic profiling. Standard metrics demonstrated that samples formed soft callus structures that later mineralized. Small molecule profiles showed distinct temporal patterns associated with the bone tissue repair process. Specifically, increased lactate and amino acid levels at early time points indicated an environment conducive to osteoblast differentiation and extracellular matrix formation. Citrate and pyruvate abundances increased at later time points indicating increasing mineral content within the defect region. Taurine, shikimate, and pantothenate distribution profiles appeared to represent a shift toward a more homeostatic remodeling environment with the differentiation and activity of osteoclasts offsetting the earlier deposition phases of bone repair. The generation of a comprehensive metabolic reference portfolio offers a potent mechanism for examining novel biomaterials and can serve as guide for the development of new targeted therapeutics to improve the rate, magnitude, and quality of bone regeneration.

Etched 3D-Printed Polycaprolactone Constructs Functionalized with Reduced Graphene Oxide for Enhanced Attachment of Dental Pulp-Derived Stem Cells.

A core challenge in the field of tissue engineering is the ability to establish pipeline workflows for the design and characterization of scaffold technologies with clinically translatable attributes. The parallel development of biomaterials and stem cell populations represents a self-sufficient and streamlined approach for establishing such a pipeline. In the current study, rat dental pulp stem cell (rDPSC) populations were established to assess functionalized polycaprolactone (PCL) constructs. Initial optimization and characterization of rDPSC extraction and culture conditions confirmed that cell populations were readily expandable and demonstrated surface markers associated with multi-potency. Subset populations were transduced to express DsRed fluorescent protein as a mechanism of tracking both cells and cell-derived extracellular matrix content on complex scaffold architecture. Thermoplastic constructs included reduced graphene oxide (rGO) as an additive to promote cellular attachment and were further modified by surface etching a weak acetic acid solution to roughen surface topographical features, which was observed to dramatically improve cell surface coverage in vitro. Based on these data, the modified rGO-functionalized PCL constructs represent a versatile platform for bone tissue engineering, capable of being applied as a standalone matrix or in conjunction with bio-active payloads such as DPSCs or other bio-inks.

Genetic profiling of human bone marrow and adipose tissue-derived mesenchymal stem cells reveals differences in osteogenic signaling mediated by graphene.

BACKGROUND: In the last decade, graphene surfaces have consistently supported osteoblast development of stem cells, holding promise as a therapeutic implant for degenerative bone diseases. However, until now no study has specifically examined the genetic changes when stem cells undergo osteogenic differentiation on graphene. RESULTS: In this study, we provide a detailed overview of gene expressions when human mesenchymal stem cells (MSCs) derived from either adipose tissue (AD-MSCs) or bone marrow (BM-MSCs), are cultured on graphene. Genetic expressions were measured using osteogenic RT2 profiler PCR arrays and compared either over time (7 or 21 days) or between each cell source at each time point. Genes were categorized as either transcriptional regulation, osteoblast-related, extracellular matrix, cellular adhesion, BMP and SMAD signaling, growth factors, or angiogenic factors. Results showed that both MSC sources cultured on low oxygen graphene surfaces achieved osteogenesis by 21 days and expressed specific osteoblast markers. However, each MSC source cultured on graphene did have genetically different responses. When compared between each other, we found that genes of BM-MSCs were robustly expressed, and more noticeable after 7 days of culturing, suggesting BM-MSCs initiate osteogenesis at an earlier time point than AD-MSCs on graphene. Additionally, we found upregulated angiogenic markers in both MSCs sources, suggesting graphene could simultaneously attract the ingrowth of blood vessels in vivo. Finally, we identified several novel targets, including distal-less homeobox 5 (DLX5) and phosphate-regulating endopeptidase homolog, X-linked (PHEX). CONCLUSIONS: Overall, this study shows that graphene genetically supports differentiation of both AD-MSCs and BM-MSCs but may involve different signaling mechanisms to achieve osteogenesis. Data further demonstrates the lack of aberrant signaling due to cell-graphene interaction, strengthening the application of specific form and concentration of graphene nanoparticles in bone tissue engineering.

A Streamlined Approach to Pathway Analysis from RNA-Sequencing Data.

The reduction in costs associated with performing RNA-sequencing has driven an increase in the application of this analytical technique; however, restrictive factors associated with this tool have now shifted from budgetary constraints to time required for data processing. The sheer scale of the raw data produced can present a formidable challenge for researchers aiming to glean vital information about samples. Though many of the companies that perform RNA-sequencing provide a basic report for the submitted samples, this may not adequately capture particular pathways of interest for sample comparisons. To further assess these data, it can therefore be necessary to utilize various enrichment and mapping software platforms to highlight specific relations. With the wide array of these software platforms available, this can also present a daunting task. The methodology described herein aims to enable researchers new to handling RNA-sequencing data with a streamlined approach to pathway analysis. Additionally, the implemented software platforms are readily available and free to utilize, making this approach viable, even for restrictive budgets. The resulting tables and nodal networks will provide valuable insight into samples and can be used to generate high-quality graphics for publications and presentations.

Examining the Characteristics and Applications of Mesenchymal, Induced Pluripotent, and Embryonic Stem Cells for Tissue Engineering Approaches across the Germ Layers.

The field of regenerative medicine utilizes a wide array of technologies and techniques for repairing and restoring function to damaged tissues. Among these, stem cells offer one of the most potent and promising biological tools to facilitate such goals. Implementation of mesenchymal stem cells (MSCs), induced pluripotent stem cells (iPSCs), and embryonic stem cells (ESCs) offer varying advantages based on availability and efficacy in the target tissue. The focus of this review is to discuss characteristics of these three subset stem cell populations and examine their utility in tissue engineering. In particular, the development of therapeutics that utilize cell-based approaches, divided by germinal layer to further assess research targeting specific tissues of the mesoderm, ectoderm, and endoderm. The combinatorial application of MSCs, iPSCs, and ESCs with natural and synthetic scaffold technologies can enhance the reparative capacity and survival of implanted cells. Continued efforts to generate more standardized approaches for these cells may provide improved study-to-study variations on implementation, thereby increasing the clinical translatability of cell-based therapeutics. Coupling clinically translatable research with commercially oriented methods offers the potential to drastically advance medical treatments for multiple diseases and injuries, improving the quality of life for many individuals.

Human Fat-Derived Mesenchymal Stem Cells Xenogenically Implanted in a Rat Model Show Enhanced New Bone Formation in Maxillary Alveolar Tooth Defects.

BACKGROUND: Due to restorative concerns, bone regenerative therapies have garnered much attention in the field of human oral/maxillofacial surgery. Current treatments using autologous and allogenic bone grafts suffer from inherent challenges, hence the ideal bone replacement therapy is yet to be found. Establishing a model by which MSCs can be placed in a clinically acceptable bone defect to promote bone healing will prove valuable to oral/maxillofacial surgeons. METHODS: Human adipose tissue-derived MSCs were seeded onto Gelfoam® and their viability, proliferation, and osteogenic differentiation was evaluated in vitro. Subsequently, the construct was implanted in a rat maxillary alveolar bone defect to assess in vivo bone healing and regeneration. RESULTS: Human MSCs were adhered, proliferated, and uniformly distributed, and underwent osteogenic differentiation on Gelfoam®, comparable with the tissue culture surface. Data confirmed that Gelfoam® could be used as a scaffold for cell attachment and a delivery vehicle to implant MSCs in vivo. Histomorphometric analyses of bones harvested from rats treated with hMSCs showed statistically significant increase in collagen/early bone formation, with cells positive for osteogenic and angiogenic markers in the defect site. This pattern was visible as early as 4 weeks post treatment. CONCLUSIONS: Xenogenically implanted human MSCs have the potential to heal an alveolar tooth defect in rats. Gelfoam®, a commonly used clinical biomaterial, can serve as a scaffold to deliver and maintain MSCs to the defect site. Translating this strategy to preclinical animal models provides hope for bone tissue engineering.

Multiomics Evaluation of Human Fat-Derived Mesenchymal Stem Cells on an Osteobiologic Nanocomposite.

Effective graft technologies for bone repair have been a primary focus in the field of bone tissue engineering. We have previously fabricated and examined a nanocomposite composed of polyurethane, nano-hydroxyapatite, and decellularized bone particles, which demonstrated osteobiologic characteristics. To evaluate the underlying mechanisms of this biomaterial, human adipose-derived mesenchymal stem cell seeded scaffolds were assessed using a combinatorial approach of transcriptomic and metabolomic analyses. Data from osteogenic and signal transduction polymerase chain reaction arrays and small molecule abundances, measured through liquid chromatography-mass spectrometry, were cross-examined using Integrated Molecular Pathway Level Analysis, Database for Annotation, Visualization, and Integrated Discovery, and ConsensusPathDB online tools to generate a fundamental collection of scaffold-influenced pathways. Results demonstrated upregulation of key osteogenic, cellular adhesion cell signaling markers and indicated that Hedgehog and Wnt signaling pathways were primary candidates for the osteobiologic mechanisms of the scaffold design. The detection of complimentary metabolites, such as ascorbate, further indicates that scaffolds generate intricate cellular environments, promoting cell attachment and subsequent osteodifferentiation.

Commercially available bone graft substitutes: the impact of origin and processing on graft functionality.

Development of effective and cost-efficient bone tissue engineering grafts has been the key area of research for regenerative medicine, yet an ideal grafting material has remained elusive due in large part to the highly dynamic nature of bone. A wide array of materials, both natural and synthetic, have been implemented as potential candidates for commercially available products, yet the gold standard for grafting material still remains autogenous bone. We review currently commercially available bone graft materials and relevant graft characteristics that impact the effectiveness of tissue repair, emphasizing the advantages and disadvantages of materials based on composition and origin. Examined materials were selected through a web-based search for readily accessible and clinically applicable graft materials. Grafts were then categorized according to material source to examine advantages and disadvantages associated with allogenic, xenogeneic, synthetic materials. Lastly, the application of bioactive molecules onto these basal grafts is explored to illustrate the enhancement and regulative capacity of these additives on traditional osteobiologic materials.

Evaluation of a Polyurethane Platform for Delivery of Nanohydroxyapatite and Decellularized Bone Particles in a Porous Three-Dimensional Scaffold.

The complex dynamic nature of bone tissue presents a unique challenge for developing optimal biomaterials within the field of bone tissue engineering. Materials based on biological and physiological characteristics of natural bone have shown promise for inducing and promoting effective bone repair. Design of multicomposite scaffolds that incorporate both malleable and hard mineral components allows for intricate structures with nano- and macrosized mineral components to provide architectural elements that promote osteogenesis. The examined S-1 and S-2 scaffolds are multilayered constructs which differ only in the compositional ratio of nanohydroxyapatite (nHA) and decellularized bone particles (DBPs). The constructs incorporated previously studied nHA/polyurethane films interspersed with macrosized bone DBPs to stimulate integration with native tissue and induce osteogenic activity. In vitro assessment of cytocompatibility and osteostimulatory characteristics indicated that the scaffolds did not negatively impact cell health and demonstrated osteogenic effects. When the constructs were implanted in vivo, in a rat tibial defect model, the biocompatibility and osteogenic impact were confirmed. Material-treated defects were observed to not induce negative tissue reactions and, in those treated with S-1 scaffolds, exhibited greater levels of new bone formation. These results indicate that, while both scaffold designs were biocompatible, S-1 constructs demonstrate more effective biologically relevant nano-/macromineral architectural elements.

Biometric Data Comparison Between Lewis and Sprague Dawley Rats.

Introduction: Pressure mapping systems are often used for indirect assessment of kinematic gait parameter differences after repair of critical peripheral nerve defects in small animal models. However, there does not appear to be any literature that studies the differences in normal gait pattern of Sprague Dawley rats compared to Lewis rats using a Tekscan VH4 pressure mat system. The purpose of this study is to assess the gait profile of Lewis and Sprague Dawley rats generated by Tekscan's VH4 system to detect similarities and/or differences in gait parameters involving both force and temporal variables. Materials and Methods: The gait profile of 14 Lewis and 14 Sprague Dawley rats was recorded using a Tekscan VH4 pressure map system with two successful walks per animal and gait parameter data was normalized for mean variance between the two rodent strains. Results: The results showed that temporal and normalized force parameters were not significantly different between the two types of rats. Maximum force, contact area, stride length, and adjusted pressure variables were significantly different between the two strains, likely attributed to the body size and weight differential between the strains. Variation in some of these parameters were considered due to differences in overall body size between the two strains, variations in gait kinematics between individual rodent subjects, and the limitations of the current experimental design. Conclusion: For future in vivo models, either Sprague Dawley or Lewis rat strains would be acceptable animal models when comparing base-line gait profiles using the Tekscan VH4 pressure map system when assessing critical defect repairs of peripheral nerves.

Polyurethane/nano-hydroxyapatite composite films as osteogenic platforms.

A wide variety of biomaterials are utilized in tissue engineering to promote cell proliferations in vitro or tissue growth in vivo. The combination of cells, extracellular matrices, and biocompatible materials may make it possible to grow functional living tissues ranging from bone to nerve cells. In bone regeneration, polymeric scaffolds can be enhanced by the addition of bioactive materials. To this end, this study designed several ratios of polyurethane (PU) and nano-hydroxyapatite (nHA) composites (PU-nHA ratios: 100/0, 90/10, 80/20, 70/30, 60/40 w/w). The physical and mechanical properties of these composites and their relative cellular compatibility in vitro were determined. The chemical composition and crystallinity of the composites were confirmed using X-ray diffraction, X-ray photoelectron spectroscopy, and thermogravimetric analyses. Atomic force microscopy, nano-indentation, and contact angle measurements were used to evaluate surface properties. The results showed a significant increase in surface roughness and a decrease in contact angle when the nHA concentration increased above 20%, resulting in a significant increase in hydrophilicity. These surface property changes influenced cellular behavior when MC 3T3-E1 cells were seeded on the composites. All composites were cytocompatible. There was a linear increase in cell proliferation on the 80/20 and 70/30 composites only, whereas subjective evaluation demonstrated noticeable clusters or nodules of cells (considered hallmarks of osteogenic differentiation) in the absence of any osteogenic inducers only on the 90/10 and 80/20 composites. Cellular data suggests that the 80/20 composite was an optimal environment for cell adhesion, proliferation, and, potentially, osteogenic differentiation in vitro.