Regenerative Potential and Inflammation-Induced Secretion Profile of Human Adipose-Derived Stromal Vascular Cells Are Influenced by Donor Variability and Prior Breast Cancer Diagnosis.

Adipose tissue contains a heterogeneous population of stromal vascular fraction (SVF) cells that work synergistically with resident cell types to enhance tissue healing. Ease of access and processing paired with therapeutic promise make SVF cells an attractive option for autologous applications in regenerative medicine. However, inherent variability in SVF cell therapeutic potential from one patient to another hinders prognosis determination for any one person. This study investigated the regenerative properties and inflammation responses of thirteen, medically diverse human donors. Using non-expanded primary lipoaspirate samples, SVF cells were assessed for robustness of several parameters integral to tissue regeneration, including yield, viability, self-renewal capacity, proliferation, differentiation potential, and immunomodulatory cytokine secretion. Each parameter was selected either for its role in regenerative potential, defined here as the ability to heal tissues through stem cell repopulation and subsequent multipotent differentiation, or for its potential role in wound healing through trophic immunomodulatory activity. These data were then analyzed for consistent and predictable patterns between and across measurements, while also investigating the influence of the donors' relevant medical histories, particularly if the donor was in remission following breast cancer treatment. Analyses identified positive correlations among the expression of three cytokines: interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1. The expression of these cytokines also positively related to self-renewal capacity. These results are potentially relevant for establishing expectations in both preclinical experiments and targeted clinical treatment strategies that use stem cells from patients with diverse medical histories.

Nanotextured titanium surfaces for enhancing skin growth on transcutaneous osseointegrated devices.

A major problem with transcutaneous osseointegrated implants is infection, mainly due to improper closure of the implant-skin interface. Therefore, the design of transcutaneous osseointegrated devices that better promote skin growth around these exit sites needs to be examined and, if successful, would clearly limit infection. Due to the success already demonstrated for orthopedic implants, developing surfaces with biologically inspired nanometer features is a design criterion that needs to be investigated for transcutaneous devices. This study therefore examined the influence of nanotextured titanium (Ti) created through electron beam evaporation and anodization on keratinocyte (skin-forming cell) function. Electron beam evaporation created Ti surfaces with nanometer features while anodization created Ti surfaces with nanotubes. Conventional Ti surfaces were largely micron rough, with few nanometer surface features. Results revealed increased keratinocyte adhesion in addition to increased keratinocyte spreading and differences in keratinocyte filopodia extension on the nanotextured Ti surfaces prepared by either electron beam evaporation or anodization compared to their conventional, unmodified counterparts after 4h. Results further revealed increased keratinocyte proliferation and cell spreading over 3 and 5days only on the nanorough Ti surfaces prepared by electron beam evaporation compared to both the anodized nanotubular and unmodified Ti surfaces. Therefore, the results from this in vitro study provided the first evidence that nano-modification techniques should be further researched as a means to possibly improve skin growth, thereby improving transcutaneous osseointegrated orthopedic implant longevity.

Recombinant bone morphogenic protein-2 in orthopaedic surgery: a review.

Bone morphogenic proteins (BMPs) are pleiotropic regulators of bone volume, skeletal organogenesis and bone regeneration after a fracture. They function as signaling agents to affect cellular events like proliferation, differentiation and extracellular matrix synthesis. Clinically utilized rhBMP-2 combines rhBMP-2 with an osteoconductive carrier to induce bone growth and acts as a bone graft substitute. rhBMP-2, initially released in 2002, has been used primarily in spinal fusions in the lumbar and cervical regions. Recently, the application of rhBMP-2 has extended into the orthopedic trauma setting with increased application in open tibia fractures. This review outlines the history of development, molecular characteristics, toxicity and clinical applications.

Sequential release of bioactive IGF-I and TGF-beta 1 from PLGA microsphere-based scaffolds.

Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.

Microencapsulated cells genetically modified to overexpress human transforming growth factor-beta1: viability and functionality in allogeneic and xenogeneic implant models.

This study explores the suitability of using encapsulated genetically modified fibroblasts for orthopedic tissue engineering by examining cell survival and persistence of human transforming growth factor-beta (hTGF-beta) overexpression in xenogeneic and allogeneic implant models. Human wild-type fibroblasts, modified to produce a latent form of hTGF-beta, and murine mutant-type fibroblasts, engineered to release a constitutively active form of hTGF-beta, were encapsulated separately in Ca2+ -alginate microcapsules. Following a percentage viability assessment by MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) test, microcapsules were implanted into either the subcutaneous or intraperitoneal cavities of mice. Explanted encapsulated cells were characterized for percentage viability and subjected to a release study and a viability test 1 week and 3 weeks following implantation, a time frame consistent with the requirement for orthopedic tissue engineering application of this growth factor. On average, percentage viabilities of encapsulated cells were 64%at implantation, 52% at explantation, and 56%after 1 week following either 1- or 3-week explantation. hTGF-beta release declined following in vivo implantation, more so for xenogeneic than allogeneic models, but remained in the clinically attractive range of 2 to 30 ng/(10(6) implanted cells 24 h). This technical platform for hTGF-beta is very encouraging for cartilage regeneration using orthopedic tissue engineering, and further evaluation is warranted.

In vitro characterization of TGF-beta1 release from genetically modified fibroblasts in Ca(2+)-alginate microcapsules.

This study was undertaken to develop an in situ source of transforming growth factor-beta1 (TGF-beta1), one of several molecules potentially useful for a tissue-engineered bioartificial cartilage. Primary human fibroblasts and murine NIH 3T3 cells were genetically modified via viral transfection to express human TGF-beta1. Two viral constructs were used, one expressing a gene encoding for the latent and the other for the constitutively active form of the growth factor. Unmodified cells served as controls. Four genetically modified cohorts and two controls were separately encapsulated in a 1.8% alginate solution using a vibrating nozzle and 0.15M calcium chloride crosslinking bath. Diameter of the spherical capsules was 410 +/- 87 microm. In vitro release rate measured over 168 hours varied with cell types and ranged from 2-17 pg/(milligram of capsules x 24 h) or 2-17 ng/(10(6) cells x 24 h). None of the formulations exhibited a large initial bolus release. Even when serum-supplemented medium was not replenished, cell viabilities remained over 55% after 1 week for all cell types. Microencapsulated genetically modified cells were capable of a constitutive synthesis and delivery of biologically significant quantity of TGF-beta1 for at least 168 hours and thus are of potential utility for artificial cartilage and other orthopedic tissue engineering applications.

Upregulation of basal TGFbeta1 levels by EMF coincident with chondrogenesis--implications for skeletal repair and tissue engineering.

Members of the TGFbeta/BMP gene family regulate cartilage and bone development. These genes are re-expressed in bone repair and are thought to mediate chondro- and osteoprogenitor cell differentiation. These observations have led to a therapeutic strategy of introducing these growth factors into experimental cartilage and bone defects. Therapeutic efficacy, however, has been limited by diffusion or inactivation of these growth factors from the desired site and by the inability to deliver sustained concentrations of growth factors. This study demonstrates an increase in basal TGFbeta mRNA and protein levels in association with chondrogenic differentiation in endochondral ossification. mRNA is increased by 158%; protein by 23%, and cells immunopositive for TGFbeta by 343% at maximal TGFbeta expression. Importantly, the pattern of TGFbeta expression is preserved throughout the developmental sequence. Our data suggest that the exposure to a specific electromagnetic field (EMF) enhances, but does not disorganize, chondrogenesis and endochondral calcification as well as the normal physiologic expression of TGFbeta. The ability to increase TGFbeta at a moderately low dose for sustained periods of time without disorganizing its physiology suggests the ability to establish temporal concentration gradients of growth factors for the purpose of stimulating skeletal repair.