Tissue engineering aims to repair, restore, and regenerate lost or damaged tissues by using biomaterials, cells, mechanical forces and factors (chemical and biological) alone or in combination. Growth factors are routinely used in the tissue engineering approach to expedite the process of regeneration. The growth factor approach has been hampered by several complications including high dose requirements, lower half-life, protein instability, higher costs and undesired side effects. Recently a variety of alternative small molecules of both natural and synthetic origin have been explored as alternatives to growth factors for tissue regeneration applications. Small molecules are simple biochemical components that elicit certain cellular responses through signaling cascades. Small molecules present a viable alternative to biological factors. Small molecule strategies can reduce various side effects, maintain bioactivity in a biological environment and minimize cost issues associated with complex biological growth factors. This manuscript focuses on three-osteoinductive small molecules, namely melatonin, resveratrol (from natural sources) and purmorphamine (synthetically designed) as inducers of bone formation and osteogenic differentiation of stem cells. Efforts have been made to summarize possible biological pathways involved in the action of each of these drugs. Melatonin is known to affect Mitogen Activated Protein (MAP) kinase, Bone morphogenic protein (BMP) and canonical wnt signaling. Resveratrol is known to activate cascades involving Wnt and NAD-dependent deacetylase sirtuin-1 (Sirt1). Purmorphamine is a Hedgehog (Hh) pathway agonist as it acts on Smoothened (Smo) receptors. These mechanisms and the way they are affected by the respective small molecules will also be discussed in the manuscript.

Scaffold based bone tissue engineering (BTE) has made great progress in regenerating lost bone tissue. Materials of natural and synthetic origin have been used for scaffold fabrication. Scaffolds derived from natural polymers offer greater bioactivity and biocompatibility with mammalian tissues to favor tissue healing, due to their similarity to native extracellular matrix (ECM) components. Often it is a challenge to fabricate natural polymer based scaffolds for BTE applications without compromising their bioactivity, while maintaining adequate mechanical properties. In this work, we report the fabrication and characterization of cellulose and collagen based micro-nano structured scaffolds using human osteoblasts (HOB) for BTE applications. These porous micro-nano structured scaffolds (average pore diameter 190 +/- 10 microm) exhibited mechanical properties in the mid range of human trabecular bone (compressive modulus 266.75 +/- 33.22 MPa and strength 12.15 3 +/- 2.23 MPa). These scaffolds supported the greater adhesion and phenotype maintenance of cultured HOB as reflected by higher levels of osteogenic enzyme alkaline phosphatase and mineral deposition compared to control polyester micro-nano structured scaffolds of identical pore properties. These natural polymer based micro-nano structured scaffolds may serve as alternatives to polyester based scaffolds for BTE applications.

Use of growth factors as biochemical molecules to elicit cellular differentiation is a common strategy in tissue engineering. However, limitations associated with growth factors, such as short half‐life, high effective physiological doses, and high costs, have prompted the search for growth factor alternatives, such as growth factor mimics and other proteins. This work explores the use of insulin protein as a biochemical factor to aid in tendon healing and differentiation of cells on a biomimetic electrospun micro‐nanostructured scaffold. Dose response studies were conducted using human mesenchymal stem cells (MSCs) in basal media supplemented with varied insulin concentrations. A dose of 100‐ng/mL insulin showed increased expression of tendon markers. Synthetic‐natural blends of various ratios of polycaprolactone (PCL) and cellulose acetate (CA) were used to fabricate micro‐nanofibers to balance physicochemical properties of the scaffolds in terms of mechanical strength, hydrophilicity, and insulin delivery. A 75:25 ratio of PCL:CA was found to be optimal in promoting cellular attachment and insulin immobilization. Insulin immobilized fiber matrices also showed increased expression of tendon phenotypic markers by MSCs similar to findings with insulin supplemented media, indicating preservation of insulin bioactivity. Insulin functionalized scaffolds may have potential applications in tendon healing and regeneration.

Rotator cuff (RC) tears represent a large proportion of musculoskeletal injuries attended to at the clinic and thereby make RC repair surgeries one of the most widely performed musculoskeletal procedures. Despite the high incidence rate of RC tears, operative treatments have provided minimal functional gains and suffer from high re-tear rates. The hypocellular nature of tendon tissue poses a limited capacity for regeneration. In recent years, great strides have been made in the area of tendonogenesis and differentiation towards tendon cells due to a greater understanding of the tendon stem cell niche, development of advanced materials, improved scaffold fabrication techniques, and delineation of the phenotype development process. Though in vitro models for tendonogenesis have shown promising results, in vivo models have been less successful. The present work investigates structured matrices mimicking the tendon microenvironment as cell delivery vehicles in a rat RC tear model. RC injuries augmented with a matrix delivering rat mesenchymal stem cells (rMSCs) showed enhanced regeneration over suture repair alone or repair with augmentation, at 6 and 12-weeks post-surgery. The local delivery of rMSCs led to increased mechanical properties and improved tissue morphology. We hypothesize that the mesenchymal stem cells function to modulate the local immune and bioactivity environment through autocrine/paracrine and/or cell homing mechanisms. This study provides evidence for improved tendon healing with biomimetic matrices and delivered MSCs with the potential for translation to larger, clinical animal models. The enhanced regenerative healing response with stem cell delivering biomimetic matrices may represent a new treatment paradigm for massive RC tendon tears.

The success of the scaffold-based bone regeneration approach critically depends on the biomaterial's mechanical and biological properties. Cellulose and its derivatives are inherently associated with exceptional strength and biocompatibility due to their β-glycosidic linkage and extensive hydrogen bonding. This polymer class has a long medical history as a dialysis membrane, wound care system and pharmaceutical excipient. Recently cellulose-based scaffolds have been developed and evaluated for a variety of tissue engineering applications. In general porous polysaccharide scaffolds in spite of many merits lack the necessary mechanical competence needed for load-bearing applications. The present study reports the fabrication and characterization of three-dimensional (3D) porous sintered microsphere scaffolds based on cellulose derivatives using a solvent/non-solvent sintering approach for load-bearing applications. These 3D scaffolds exhibited a compressive modulus and strength in the mid-range of human trabecular bone and underwent degradation resulting in a weight loss of 10-15% after 24 weeks. A typical stress-strain curve for these scaffolds showed an initial elastic region and a less-stiff post-yield region similar to that of native bone. Human osteoblasts cultured on these scaffolds showed progressive growth with time and maintained expression of osteoblast phenotype markers. Further, the elevated expression of alkaline phosphatase and mineralization at early time points as compared to heat-sintered poly(lactic acid-glycolic acid) control scaffolds with identical pore properties affirmed the advantages of polysaccharides and their potential for scaffold-based bone regeneration.

Scaffold architecture, surface topography, biochemical and mechanical cues have been shown to significantly improve cellular events and in vivo tissue regeneration. Specifically electrospun nanofiber matrices have gained tremendous interest due to their intrinsic structural resemblance to native tissue extracellular matrix (ECM). The present study reports on the electrospun nanofiber matrices of polycaprolactone (PCL)-chitosan (CS) blends and effect of type I collagen surface functionalization in regulating rat bone marrow derived stromal cells (rBMSCs) differentiation into osteogenic lineage. Collagen was covalently attached to blend nanofibers via carbodiimide (EDC) coupling. Bead-free smooth nanofibers (diameter-700-850 nm) obtained at the optimized conditions of polymer concentration and electrospinning parameters were used for the study. EDC collagen coupling resulted in 0.120+/-0.016 micro g of collagen immobilization onto a 1 cm2 area of the PCL/CS nanofibers, which was 2.6-folds higher than the amount of collagen that can be retained by physical adsorption. Significantly improved rBMSCs adhesion, spreading, proliferation and osteogenic differentiation was observed on the collagen functionalized COL-PCULCS nanofiber matrices as compared to control groups. Osteogenic phenotypic markers such as alkaline phosphatase (ALP) activity and mineralization were found to be significantly higher on COL-PCL/CS nanofiber matrices as compared to controls. Elevated gene expression profiles of osteogenic markers such as osteocalcin (0CN), osteopontin (OPN) and ALP further corroborate the osteoinductive nature of the collagen functionalized PCL/CS nanofiber matrices. These fiber matrices and modification techniques could be extended to other scaffold systems for tissue engineering applications.

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Electrospun nanofiber matrices have attracted a great deal of attention as matrices for skin repair and regeneration. The current manuscript reports the fabrication and characterization of a bioactive polycaprolactone (PCL) fiber matrix for its ability to deliver multiple factors. Bioactive PCL matrices were created by incorporating a model angiogenic factor and a model antibiotic drug. Chitosan coating on the fiber matrices significantly improved the ability to hold moisture and contributed to antibiotic activity. These fiber matrices have a modulus of 5.8 6 1.3 MPa and matrices subjected to degradation over 4 weeks did not lose their tensile properties due to slow degradation rate. Chitosan coating avoided the initial burst release commonly associated with fiber matrices and only 60% of the encapsulated drug was released over a period of 15 days. Control PCL-chitosan matrices were able to reduce Staphylococcus aureus (S. aureus) growth both in static and dynamic condition as compared to formulations with 50 mg gentamicin. In general, all the fiber matrices were able to support fibroblast growth and maintained normal cell morphology. Such bioactive bandages may serve as versatile and less expensive alternatives for the treatment of complex wounds.