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Stimulation of bone regeneration using growth factors is a promising approach for musculoskeletal regenerative engineering. Common limitations with protein growth factors are high manufacturing costs, protein instability, contamination issues, and unwanted immunogenic responses of the host. New strategies for bone regeneration that obviate these problems can have a significant impact on the treatment of skeletal injury and diseases. Over the past decade, a large number of small molecules with the potential of regenerating skeletal tissue have been reported in the literature. Here, we review this literature, paying specific attention to the prospects for small molecule-based bone-regenerative engineering. We also review the preclinical study of small molecules associated with bone regeneration.

The uses of bone morphogenetic proteins and parathyroid hormone therapeutics are fraught with several fundamental problems, such as cost, protein stability, immunogenicity, contamination and supraphysiological dosage. These downsides may effectively limit their more universal use. Therefore, there is a clear need for alternative forms of biofactors to obviate the drawbacks of protein-based inductive factors for bone repair and regeneration. Our group has studied small molecules with the capacity to regulate osteoblast differentiation and mineralization because their inherent physical properties minimize limitations observed in protein growth factors. For instance, in general, small molecule inducers are usually more stable, highly soluble, nonimmunogenic, more affordable and require lower dosages. Small molecules with the ability to induce osteoblastic differentiation may represent the next generation of bone regenerative medicine. This review describes efforts to develop small molecule-based biofactors for induction, paying specific attention to their novel roles in bone regeneration.

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Osteoblastic differentiation is an important landmark for bone formation, bone repair and regeneration, however it is a very complex process controlled by different signaling mechanisms. Several groups have reported that the cyclic adenosine monophosphate (cAMP) signaling system is responsible for regulating osteoblast cell differentiation. Nonetheless, to date, the principle role of the cAMP molecules related to this process remains controversial. Moreover, the underlying cAMP-dependent signaling cascade governing the osteoblastic differentiation has not been clarified. In this report, we investigated the roles of the cAMP-dependent protein kinase A (PKA) signaling in proliferation, differentiation and mineralization of osteoblast-like MC3T3-E1 cells using the PKA-specific small molecule cAMP analogue, 6-Bnz-cAMP, at 100 µM. Alkaline phosphatase (ALP) activity, runt transcription factor 2 (Runx2), osteopontin (OPN) and osteocalcin (OCN) protein expressions were used as osteoblast-specific markers to demonstrate osteoblastic differentiation. Further, calcium measurement of the extracellular matrix was employed as the hallmark of matrix mineralization or calcification. We report here that activation of PKA by the small molecule 6-Bnz-cAMP induces osteoblastic differentiation and matrix mineralization of osteoblast-like MC3T3-E1 cells. Moreover, 6-Bnz-cAMP does not induce cytotoxicity to the cells as revealed by our cell proliferation studies. Therefore, based on these findings, we propose that the PKA-specific small molecule 6-Bnz-cAMP may serve as a novel bone-inducing growth factor for repairing and regenerating bone tissues during bone regenerative engineering.

Cytoplasmic dynein 1 2 is a motor complex responsible for the transport of membranous vesicles and different cargo proteins toward the minus ends of microtubules including mitosis, nuclear migration, Golgi and centrosome localization, organelle and viral transport, and axonal transport (1-4). It is a large multisubunit assembly (ϳ1.5 MDa) containing two copies each of DYNC1H1 (the heavy chain), DYNC1I (the intermediate chains), DYNC1LI (the light intermediate chains), and two copies of each of three distinct light chain families (1, 5, 6). The globular heads of the cytoplasmic dynein heavy chains generate the force for microtubule-based motility (7-9). The heavy chains dimerize via their N-terminal stalks, and the stalks contain both the light intermediate chain and the intermediate chain binding regions (10, 11).The intermediate chains, light intermediate chains, and three pairs of light chains are at the base of the cytoplasmic dynein 1 complex and form its cargo binding domain (2, 12-15). The two intermediate chains are essential components of the cytoplasmic dynein complex, serving as platforms that interact with the heavy chains, the three light chain dimers, as well as the p150 subunit of dynactin, a cargo adaptor complex, and other protein cargoes (12,13,16,17). The C-terminal half of the protein contains seven WD repeats (18,19), which are presumed to form a ␤ propeller structure responsible for interacting with the heavy chain (4,20). The N terminus of the intermediate chain is predicted to form a coiled-coil domain with six heptad repeats that may be involved in intra-or interchain protein interactions (21), including the interaction with the p150 subunit of dynactin (12). The distinct interaction domains for the three light chain families, DYNLL (LC8), DYNLT (Tctex1), and DYNLRB (Roadblock), are also in the N-terminal half of the intermediate chain (22)(23)(24).Biochemical analyses showed that cytoplasmic dynein 1 can be fractionated into two complexes, one containing the heavy chains and the light intermediate chains and the second composed of the intermediate chains and three light chain families (25-27), but it is not known how the intermediate chains might interact directly or whether their interaction requires one or more of the light chain families. A role for the LC8 light chain as a molecular glue has been suggested by its presence in complexes unrelated to dynein (3,28). Supporting this hypothesis are the observations that an N-terminal fragment of the intermediate chain becomes more ordered upon binding of the LC8 or Tctex1 light chains (29,30). In addition, there are two regions of alternative splicing in the N terminus of the two intermediate chain genes that generate at least six unique isoforms (1,12,21,31,32). Although the functional significance of these intermediate chain isoforms is unknown, there are data supporting the hypothesis that the isoforms of the light intermediate chains and isoforms of the light chain families contribute to the specificity of dynein cargo binding, reviewe...

Osteoblast cell adhesion and differentiation on biomaterials are important achievements necessary for implants to be useful in bone regenerative engineering. Recombinant bone morphogenetic proteins (BMPs) have been shown to be important for these processes; however, there are many challenges associated with the widespread use of these proteins. A recent report demonstrated that the small molecule phenamil, a diuretic derivative, was able to induce osteoblast differentiation and mineralization in vitro via the canonical BMP signalling cascade (Park et al., 2009). In this study, the feasibility of using phenamil as a novel biofactor in conjunction with a biodegradable poly(lactide-co-glycolide acid) (PLAGA) polymeric scaffold for engineering bone tissue was evaluated. The in vitro cellular behaviour of osteoblast-like MC3T3-E1 cells cultured on PLAGA scaffolds in the presence of phenamil at 10 μM were characterized with regard to initial cell adhesion, proliferation, alkaline phosphatase (ALP) activity and matrix mineralization. The results demonstrate that phenamil supported cell proliferation, promoted ALP activity and facilitated matrix mineralization of osteoblast-like MC3T3-E1 cells. Moreover, in this study, we found that phenamil promoted integrin-mediated cell adhesion on PLAGA scaffolds. It was also shown that phenamil encapsulated within porous, microsphere PLAGA scaffolds retained its osteogenic activity upon release. Based on these findings, the small molecule phenamil has the potential to serve as a novel biofactor for the repair and regeneration of bone tissues.