Faculty Bibliography

BACKGROUND: : The graying of our population has motivated the authors to better understand age-related impairments in wound healing. To increase research throughput, the authors hypothesized that the Hutchinson-Gilford progeria syndrome Zmpste24-deficient (Zmpste24) mouse could serve as a model of senescent wound healing. METHODS: : Using a stented excisional wound closure model, the authors tested this hypothesis on 8-week-old male Zmpste24 mice (n = 25) and age-matched male C57BL/6J wild-type mice (n = 25). Wounds were measured photogrammetrically and harvested for immunohistochemistry, enzyme-linked immunosorbent assay, and quantitative real-time polymerase chain reaction, and circulating vasculogenic progenitor cells were measured by flow cytometry. RESULTS: : Zmpste24 mice had a significant delay in wound closure compared with wild-type mice during the proliferative/vasculogenic phase. Zmpste24 wounds had decreased proliferation, increased 8-hydroxy-2'-deoxyguanosine levels, increased proapoptotic signaling (i.e., p53, PUMA, BAX), decreased antiapoptotic signaling (i.e., Bcl-2), and increased DNA fragmentation. These changes correlated with decreased local vasculogenic growth factor expression, decreased mobilization of bone marrow-derived vasculogenic progenitor cells, and decreased new blood vessel formation. Age-related impairments in wound closure are multifactorial. CONCLUSIONS: : The authors' data suggest that the Hutchinson-Gilford progeria syndrome Zmpste24 progeroid syndrome shares mechanistic overlap with normal aging and therefore might provide a uniquely informative model with which to study age-associated impairments in wound closure.

Encased in lacunae, osteocytes receive nutrition and biomechanical signals through the lacunocanalicular system. We have developed a novel flow-perfusion bioreactor designed to support lacunocanalicular fluid flow. We hypothesize that ex vivo fluid flow can maintain endochondral bone viability and, ultimately, serve as a novel model to study bone biology in vitro. Sprague-Dawley rat femurs were harvested, stripped of soft tissue, loaded into a custom-designed bioreactor and perfused with osteogenic culture medium. After 14 days of flow-perfusion or static culture, the bones were harvested, fixed, decalcified, embedded, sectioned and stained with haematoxylin and eosin. Fresh long bone samples were similarly processed for comparison. Osteocyte viability and function were also evaluated, using thiazolyl blue tetrazolium bromide (MTT), fluorospectrophotometric DNA quantification, alkaline phosphatase (ALP) colorimetric assay and fluorochrome labelling of mineralizing surfaces. All samples remained free of infection throughout the study period. After 14 days of flow perfusion, histological analysis showed normal-appearing bony architecture, with 72% of lacunae being osteocyte-filled compared with 93% in freshly harvested samples and only 36% in static samples. MTT staining and assay confirmed osteocyte viability in the flow-perfusion samples as well as in fresh samples. DNA quantification demonstrated DNA to be preserved in flow-perfused samples when compared with freshly harvested samples. ALP activity in flow-perfusion explants was upregulated compared with fresh and static samples. Fluorochrome-labelled mineralizing surfaces were seen throughout the explanted flow-perfused samples. This is the first demonstration that flow perfusion provides adequate chemotransportation to explanted murine endochondal bones

A serious consequence of diabetes mellitus is impaired wound healing, which largely resists treatment. We previously reported that topical application of calreticulin (CRT), an endoplasmic reticulum chaperone protein, markedly enhanced the rate and quality of wound healing in an experimental porcine model of cutaneous repair. Consistent with these in vivo effects, in vitro CRT induced the migration and proliferation of normal human cells critical to the wound healing process. These functions are particularly deficient in poor healing diabetic wounds. Using a genetically engineered diabetic mouse (db/db) in a full-thickness excisional wound healing model, we now show that topical application of CRT induces a statistically significant decrease in the time to complete wound closure compared with untreated wounds by 5.6 days (17.6 vs. 23.2). Quantitative analysis of the wounds shows that CRT increases the rate of reepithelialization at days 7 and 10 and increases the amount of granulation tissue at day 7 persisting to day 14. Furthermore, CRT treatment induces the regrowth of pigmented hair follicles observed on day 28. In vitro, fibroblasts isolated from diabetic compared with wild-type mouse skin and human fibroblasts cultured under hyperglycemic compared with normal glucose conditions proliferate and strongly migrate in response to CRT compared with untreated controls. The in vitro effects of CRT on these functions are consistent with CRT's potent effects on wound healing in the diabetic mouse. These studies implicate CRT as a potential powerful topical therapeutic agent for the treatment of diabetic and other chronic wounds.

Bone repair and regeneration is a dynamic process that involves a complex interplay between the (1) ground substance, (2) cells, and (3) milieu. While each constituent is integral to the final product, it is often helpful to consider each component individually. Therefore, we created a two-part review to examine scaffolds and cells' roles in bone tissue engineering. In Part I, we review the myriad of materials use for in vivo bone engineering. In Part II, we discuss the variety cell types (e.g., osteocytes, osteoblasts, osteoclasts, chondrocytes, mesenchymal stem cells, and vasculogenic cells) that are seeded upon or recruited to these scaffolds. In Part III, we discuss the optimization of the microenvironment. The biochemical processes and sequence of events that guide matrix production, cellular activation, and ossification are vital to developing successful bone tissue engineering strategies and are thus succinctly reviewed herein.

BACKGROUND: : Lipoaspirate centrifugation creates graded density of adipose tissue. High-density fat contains more vasculogenic cytokines and progenitor cells and has greater graft survival than low-density fat. The authors hypothesize that accelerating the bone marrow-derived progenitor cell response to injected low-density fat will improve its graft survival. METHODS: : Male 8-week-old FVB mice (n = 60) were grafted with either high-density (n = 20) or low-density (n = 40) human lipoaspirate. Half of the mice receiving low-density fat (n = 20) were treated with a stem cell mobilizer for 14 days. Grafted fat was harvested at 2 and 10 weeks for analysis. RESULTS: : Low-density fat, low-density fat plus daily AMD3100, and high-density fat had 26 +/- 3.0, 61.2 +/- 7.5, and 49.6 +/- 3.5 percent graft survival, respectively, at 2 weeks (low-density fat versus low-density fat plus daily AMD3100 and low-density fat versus high-density fat, both p < 0.01). Similar results were observed 10 weeks after grafting. Mice receiving low-density fat plus daily AMD3100 had significantly more vasculogenic progenitor cells per cubic centimeter of peripheral blood (p < 0.01) and more new blood vessels (p < 0.01). Both low-density fat plus daily AMD3100 and high-density fat contained more stromal-derived factor-1alpha and vascular endothelial growth factor mRNA/protein. CONCLUSION: : Endogenous progenitor cell mobilization enhances low-density fat neovascularization, increases vasculogenic cytokine expression, and improves graft survival to a level equal to that of high-density fat grafts.

Bone repair and regeneration is a dynamic process that involves a complex interplay between the (1) ground substance; (2) cells; and (3) milieu. Each constituent is integral to the final product, but it is often helpful to consider each component individually. While bone tissue engineering has capitalized on a number of breakthrough technologies, one of the most valued advancements is the incorporation of mesenchymal stem cells (SCs) into bone tissue engineering applications. With this new idea, however, came new found problems of guiding SC differentiation. Moreover, investigators are still working to understand which SCs source produces optimal bone formation in vitro and in vivo. Bone marrow-derived mesenchymal SCs and adipose-derived SCs have been researched most extensively, but other SC sources, including dental pulp, blood, umbilical cord blood, epithelial cells reprogrammed to become induced pluripotent SCs, among others, are being investigated. In Part II of this review series, we discuss the variety of cell types (e.g., osteocytes, osteoblasts, osteoclasts, chondrocytes, mesenchymal SCs, and vasculogenic cells) important in bone tissue engineering.

Since obesity impairs wound healing and bone marrow (BM)-derived vasculogenic progenitor cells (PCs) are important for tissue repair, we hypothesize that obesity-impaired wound healing is due, in part, to impaired PC mobilization, trafficking, and function. Peripheral blood was obtained from nondiabetic, obese (BMI > 30, n = 25), and nonobese (BMI < 30, n = 17) subjects. Peripheral blood human (h)PCs were isolated, quantified, and functionally assessed. To corroborate the human experiments, 6-mm stented wounds were created on nondiabetic obese mice (TALLYHO/JngJ, n = 15) and nonobese mice (SWR/J, n = 15). Peripheral blood mouse (m)PCs were quantified and wounds were analyzed. There was no difference in the number of baseline circulating hPCs in nondiabetic, obese (hPC-ob), and nonobese (hPC-nl) subjects, but hPC-ob had impaired adhesion (p < 0.05), migration (p < 0.01), and proliferation (p < 0.001). Nondiabetic obese mice had a significant decrease in the number of circulating PCs (mPC-ob) at 7 (p = 0.008) and 14 days (p = 0.003) after wounding. The impaired circulating mPC-ob response correlated with significantly impaired wound closure at days 14 (p < 0.001) and 21 (p < 0.001) as well as significantly fewer new blood vessels in the wounds (p < 0.001). Our results suggest that obesity impairs the BM-derived vasculogenic PC response to peripheral injury and this, in turn, impairs wound closure.

ABSTRACT: Microporous scaffolds designed to improve bony repair have had limited success; therefore, we sought to evaluate whether time-released porous scaffolds with or without recombinant bone morphogenetic protein 2 (rhBMP-2) could enhance stem cell osteoinduction. Custom-made 15/85 hydroxyapatite/beta-tricalcium phosphate scaffolds were left empty (E) or filled with rhBMP-2 (E+), calcium sulfate (CS), or CS and rhBMP-2 (CS+). All scaffolds were placed in media and weighed daily. Conditioned supernatant was analyzed for rhBMP-2 and then used to feed human adipose-derived mesenchymal stem cells (ASCs). Adipose-derived mesenchymal stem cell ALP activity, OSTERIX expression, and bone nodule formation were determined. E scaffolds retained 97% (SD, 2%) of the initial weight, whereas CS scaffolds had a near-linear 30% (SD, 3%) decrease over 60 days. E+ scaffolds released 155 (SD, 5) ng of rhBMP-2 (77%) by day 2. In contrast, CS+ scaffolds released only 30 (SD, 2) ng (10%) by day 2, and the remaining rhBMP-2 was released over 20 days. Conditioned media from E+ scaffolds stimulated the highest ALP activity and OSTERIX expression in ACSs on day 2. However, after day 6, media from CS+ scaffolds stimulated the highest ALP activity and OSTERIX expression in ASCs. Adipose-derived mesenchymal stem cells exposed to day 8 CS+-conditioned media produced significantly more bone nodules (10.1 [SD, 1.7] nodules per high-power field) than all other scaffolds. Interestingly, day 8 conditioned media from CS scaffolds simulated significantly more bone nodules than either E or E+ scaffold (P < 0.05 for both). Time-released hydroxyapatite/beta-tricalcium phosphate porosity provides sustained growth factor release, enhances ASC osteoinduction, and may result in better in vivo bone formation.