Jeremy B. Vines

Osteogenic differentiation of human mesenchymal stem cells synergistically enhanced by biomimetic peptide amphiphiles combined with conditioned medium.

An attractive strategy for bone tissue engineering is the use of extracellular matrix (ECM) analogous biomaterials capable of governing biological response based on synthetic cell-ECM interactions. In this study, peptide amphiphiles (PAs) were investigated as an ECM-mimicking biomaterial to provide an instructive microenvironment for human mesenchymal stem cells (hMSCs) in an effort to guide osteogenic differentiation. PAs were biologically functionalized with ECM isolated ligand sequences (i.e. RGDS, DGEA), and the osteoinductive potential was studied with or without conditioned medium, containing the supplemental factors of dexamethasone, β-glycerol phosphate and ascorbic acid. It was hypothesized that the ligand-functionalized PAs would synergistically enhance osteogenic differentiation in combination with conditioned medium. Concurrently, comparative evaluations independent of osteogenic supplements investigated the differentiating potential of the functionalized PA scaffolds as promoted exclusively by the inscribed ligand signals, thus offering the potential for therapeutic effectiveness under physiological conditions. Osteoinductivity was assessed by histochemical staining for alkaline phosphatase (ALP) and quantitative real-time polymerase chain reaction analysis of key osteogenic markers. Both of the ligand-functionalized PAs were found to synergistically enhance the level of visualized ALP activity and osteogenic gene expression compared to the control surfaces lacking biofunctionality. Guided osteoinduction was also observed without supplemental aid on the PA scaffolds, but at a delayed response and not to the same phenotypic levels. Thus, the biomimetic PAs foster a symbiotic enhancement of osteogenic differentiation, demonstrating the potential of ligand-functionalized biomaterials for future bone tissue repair.

Biphasic peptide amphiphile nanomatrix embedded with hydroxyapatite nanoparticles for stimulated osteoinductive response.

Formation of the native bone extracellular matrix (ECM) provides an attractive template for bone tissue engineering. The structural support and biological complexity of bone ECM are provided within a composite microenvironment that consists of an organic fibrous network reinforced by inorganic hydroxyapatite (HA) nanoparticles. Recreating this biphasic assembly, a bone ECM analogous scaffold comprising self-assembling peptide amphiphile (PA) nanofibers and interspersed HA nanoparticles was investigated. PAs were endowed with biomolecular ligand signaling using a synthetically inscribed peptide sequence (i.e., RGDS) and integrated with HA nanoparticles to form a biphasic nanomatrix hydrogel. It was hypothesized the biphasic hydrogel would induce osteogenic differentiation of human mesenchymal stem cells (hMSCs) and improve bone healing as mediated by RGDS ligand signaling within PA nanofibers and embedded HA mineralization source. Viscoelastic stability of the biphasic PA hydrogels was evaluated with different weight concentrations of HA for improved gelation. After demonstrating initial viability, long-term cellularity and osteoinduction of encapsulated hMSCs in different PA hydrogels were studied in vitro. Temporal progression of osteogenic maturation was assessed by gene expression of key markers. A preliminary animal study demonstrated bone healing capacity of the biphasic PA nanomatrix under physiological conditions using a critical size femoral defect rat model. The combination of RGDS ligand signaling and HA nanoparticles within the biphasic PA nanomatrix hydrogel demonstrated the most effective osteoinduction and comparative bone healing response. Therefore, the biphasic PA nanomatrix establishes a well-organized scaffold with increased similarity to natural bone ECM with the prospect for improved bone tissue regeneration.

Adult stem cells and tissue engineering strategies for salivary gland regeneration: a review

Saliva is an important compound produced by the salivary glands and performs numerous functions. Hyposalivation (dry mouth syndrome) is a deleterious condition often resulting from radiotherapy for patients with head and neck cancer, Sjogren’s Syndrome, or as a side effect of certain medications. Hyposalivation negatively affects speaking, mastication, and swallowing in afflicted patients, greatly reducing their quality of life. Current treatments for this pathology include modifying lifestyle, synthetic saliva supplementation, and the utilization of salivary gland stimulants and sialagogues. However, many of these treatments do not address the underlying issues and others are pervaded by numerous side effects. In order to address the shortcomings related to current treatment modalities, many groups have diverted their attention to utilizing tissue engineering and regenerative medicine approaches. Tissue engineering is defined as the application of life sciences and materials engineering toward the development of tissue substitutes that are capable of mimicking the structure and function of their natural analogues within the body. The general underlying strategy behind the development of tissue engineered organ substitutes is the utilization of a combination of cells, biomaterials, and biochemical cues intended to recreate the natural organ environment. The purpose of this review is to highlight current bioengineering approaches for salivary gland tissue engineering and the adult stem cell sources used for this purpose. Additionally, future considerations in regard to salivary gland tissue engineering strategies are discussed.

Hydroxyapatite nanoparticle reinforced peptide amphiphile nanomatrix enhances the osteogenic differentiation of mesenchymal stem cells by compositional ratios

In the field of bone tissue engineering, there is a need for materials that mimic the native bone extracellular matrix (ECM). This need is met through the creation of biphasic composites intended to mimic both the organic and inorganic facets of the native bone ECM. However, few studies have created composites with organic ECM analogous components capable of directing cellular behaviors and many are not fabricated in the nanoscale. Furthermore, few attempts have been made at investigating how variations of organic and inorganic components affect the osteogenic differentiation of human mesenchymal stem cells (hMSCs). To address these issues, biphasic nanomatrix composites consisting of hydroxyapatite nanoparticles (HANPs) embedded within peptide amphiphile (PA) nanofibers tailored with the RGDS cellular adhesion motif (PA-RGDS) were created at various HANP to PA-RGDS ratios. Fabrication of these biphasic nanomatrix composites was confirmed via scanning electron microscopy (SEM) and transmission electron microscopy (TEM). The long-term cellularity and osteogenic differentiation of hMSCs in response to the different compositional ratios were then assessed by quantifying the timed expression of genes indicative of osteogenic differentiation, alkaline phosphatase activity, and DNA content over time. Decreased cellularity and the expression of genes over time correlated with increasing compositional ratios between HANP and PA-RGDS. The highest HANP to PA-RGDS ratio (66% HANP) exhibited the greatest improvement to the osteogenic differentiation of hMSCs. Overall, these results demonstrate that the compositional ratio of biphasic nanomatrix composites plays an important role in influencing the osteogenic differentiation of hMSCs. Based on the observations presented within this study, these biphasic nanomatrix composites show promise for future usage in bone tissue engineering applications.

Cryopreserved Amniotic Suspension for the Treatment of Knee Osteoarthritis

There are few treatment options for symptomatic knee osteoarthritis (OA). Human amniotic suspension allografts (ASA) have anti-inflammatory and chondroregenerative potential and thus represent a promising treatment strategy. In anticipation of a large, placebo-controlled trial of intra-articular ASA for symptomatic knee OA, an open-label prospective feasibility study was performed. Six patients with Kellgren-Lawrence grades 3 and 4 tibiofemoral knee OA were administered a single intra-articular ASA injection containing cryopreserved particulated human amnion and amniotic fluid cells. Patients were followed for 12 months after treatment. No significant injection reactions were noted. Compared with baseline there were (1) no significant effect of the ASA injection on blood cell counts, lymphocyte subsets, or inflammatory markers and (2) a small, but statistically significant increase in serum IgG and IgE levels. Patient-reported outcomes including International Knee Documentation Committee, Knee Injury and Osteoarthritis Outcome, and Single Assessment Numeric Evaluation scores were collected throughout the study and evaluated for up to 12 months. Overall, this study demonstrates the feasibility of a single intra-articular injection of ASA for the treatment of knee OA and provides the foundation for a large placebo-controlled trial of intra-articular ASA for symptomatic knee OA.

Improved MIN6 β-Cell Function on Self-Assembled Peptide Amphiphile Nanomatrix Inscribed with Extracellular Matrix-Derived Cell Adhesive Ligands†

Understanding the role of the pancreatic extracellular matrix (ECM) in supporting islet survival and function drives the pursuit to create biomaterials that imitate and restore the pancreatic ECM microenvironment. To create an ECM mimic holding bioinductive cues for β-cells, self-assembled peptide amphiphiles (PAs) inscribed with four selected ECM-derived cell adhesive ligands are synthesized. After 7 days, compared to control groups cultured on biologically inert substrates, MIN6 β-cells cultured on PAs functionalized with YIGSR and RGDS cell adhesive ligands exhibit elevated insulin secretion in responses to glucose and also form β-cell clusters. These findings suggest that the self-assembled PA nanomatrix may be utilized to improve pancreatic islet transplantation for treating type 1 diabetes.

Enhanced MIN-6 beta cell survival and function on a nitric oxide-releasing peptide amphiphile nanomatrix

Innovative biomaterial strategies are required to improve islet cell retention, viability, and functionality, and thereby obtain clinically successful outcomes from pancreatic islet cell transplantation. To address this need, we have developed a peptide amphiphile-based nanomatrix that incorporates multifunctional bioactive cues and sustained release of nitric oxide. The goal of this study was to evaluate the effect of this peptide amphiphile nanomatrix on the viability and functionality of MIN-6 islet cells. Additionally, this study provides insight into the role of nitric oxide in islet cell biology, given that conventional nitric oxide donors are unable to release nitric oxide in a controlled, sustained manner, leading to ambiguous results. It was hypothesized that controlled nitric oxide release in synergy with multifunctional bioactive cues would promote islet cell viability and functionality. Nitric oxide-releasing peptide amphiphile nanomatrices within the range of 16.25 μmol to 130 μmol were used to analyze MIN-6 cell behavior. Both 32.5 μmol and 65 μmol peptide amphiphiles showed improved MIN-6 functionality in response to glucose over a 7-day time period, and the elevated functionality was correlated with both PDX-1 and insulin gene expression. Our results demonstrate that nitric oxide has a beneficial effect on MIN-6 cells in a concentration-dependent manner.

Angiogenic and Osteogenic Synergy of Human Mesenchymal Stem Cells and Human Umbilical Vein Endothelial Cells Cocultured on a Nanomatrix

To date, bone tissue regeneration strategies lack an approach that effectively provides an osteogenic and angiogenic environment conducive to bone growth. In the current study, we evaluated the osteogenic and angiogenic response of human mesenchymal stem cells (hMSCs) and green fluorescent protein-expressing human umbilical vein endothelial cells (GFP-HUVECs) cocultured on a self-assembled, peptide amphiphile nanomatrix functionalized with the cell adhesive ligand RGDS (PA-RGDS). Analysis of alkaline phosphatase activity, von Kossa staining, Alizarin Red quantification, and osteogenic gene expression, indicates a significant synergistic effect between the PA-RGDS nanomatrix and coculture that promoted hMSC osteogenesis. In addition, coculturing on PA-RGDS resulted in enhanced HUVEC network formation and upregulated vascular endothelial growth factor gene and protein expression. Though PA-RGDS and coculturing hMSCs with HUVECs were each previously reported to individually enhance hMSC osteogenesis, this study is the first to demonstrate a synergistic promotion of HUVEC angiogenesis and hMSC osteogenesis by integrating coculturing with the PA-RGDS nanomatrix. We believe that using the combination of hMSC/HUVEC coculture and PA-RGDS substrate is an efficient method for promoting osteogenesis and angiogenesis, which has immense potential as an efficacious, engineered platform for bone tissue regeneration.

A percutaneous, minimally invasive annulus fibrosus needle puncture model of intervertebral disc degeneration in rabbits:

Purpose: Various animal models have been proposed to mimic the pathophysiologic process of intervertebral disc degeneration, a leading cause of back pain. The purpose of this study is to describe a minimally invasive technique via percutaneous needle puncture of the annulus fibrosus in New Zealand white rabbits. Methods: Under fluoroscopic guidance, an 18-gauge spinal needle was inserted 2 cm lateral to the midline spinous process. The needle was slowly advanced at approximately 45° angle until it was adjacent to the L5/L6 disc space. Lateral and anteroposterior views were used to verify correct needle position before advancing into the nucleus pulposus. The rabbits underwent weekly X-rays for 4 weeks to assess disc height index. MRI T2 relaxation was evaluated at week four to assess morphological changes. Discs were histologically graded on a 12-point scale to assess degeneration and compared to discs obtained from uninjured rabbits. Results: There were no complications associated with the percutaneous needle puncture procedure. All animals survived the duration of the experiment. Four weeks after injury, the disc height had progressively narrowed to approximately 50% of baseline. MRI assessment at the 4-week time point demonstrated a mean T2 relaxation time at the L5/L6 level that was 20.9% of the T2 relaxation time at the uninjured L4/L5 disc level (p < 0.001). Histological analysis demonstrated lamellar disorganization of the annulus and decreased cellularity and proteoglycan content within the injured nucleus compared to uninjured control discs. Conclusion: The present study demonstrated a reliable technique of inducing an annular tear via a percutaneous needle puncture. Compared to open surgical approaches, the percutaneous model produces similar progressive disc degeneration while minimizing harm to the animal subjects. Clinical Relevance: The present study establishes a technique for the introduction of novel therapeutic agents to treat disc degeneration that may translate to future clinical trials.

Novel Multifunctional Nanomatrix Reduces Inflammation in Dynamic Conditions in Vitro and Dilates Arteries ex Vivo

Inflammatory responses play a critical role in tissue-implant interactions, often limiting current implant utility. This is particularly true for cardiovascular devices. Existing stent technology does little to avoid or mitigate inflammation or to influence the vasomotion of the artery after implantation. We have developed a novel endothelium-mimicking nanomatrix composed of peptide amphiphiles that enhances endothelialization while decreasing both smooth muscle cell proliferation and platelet adhesion. Here, we evaluated whether the nanomatrix could prevent inflammatory responses under static and physiological flow conditions. We found that the nanomatrix reduced monocyte adhesion to endothelial cells and expression of monocyte inflammatory genes (TNF-α, MCP-1, IL-1β, and IL-6). Furthermore, the nitric-oxide releasing nanomatrix dramatically attenuated TNF-α-stimulated inflammatory responses as demonstrated by significantly reduced monocyte adhesion and inflammatory gene expression in both static and physiological flow conditions. These effects were abolished by addition of a nitric oxide scavenger. Finally, the nanomatrix stimulated vasodilation in intact rat mesenteric arterioles after constriction with phenylephrine, demonstrating the bioavailability and bioactivity of the nanomatrix, as well as exhibiting highly desired release kinetics. These results demonstrate the clinical potential of this nanomatrix by both preventing inflammatory responses and promoting vasodilation, critical improvements in stent and cardiovascular device technology.