Type I and II Diabetic Adipose-Derived Stem Cells Respond In Vitro to Dehydrated Human Amnion/Chorion Membrane Allograft Treatment by Increasing Proliferation, Migration, and Altering Cytokine Secretion.

Objective: Human amniotic membranes have been shown to be effective for healing diabetic foot ulcers clinically and to regulate stem cell activity in vitro and in vivo; however, diabetic stem cells may be impaired as a sequela of the disease. In this study, dehydrated human amnion/chorion membrane (dHACM) allografts (EpiFix®; MiMedx Group) were evaluated for their ability to regulate diabetic stem cells in vitro. Approach: Human adipose-derived stem cells (ADSCs) from normal, type I diabetic, and type II diabetic donors were treated with soluble extracts of dHACM and evaluated for proliferation after 3 days by DNA assay, chemotactic migration after 1 day by transwell assay, cytokine secretion after 3 days by multiplex ELISA, and gene expression after 5 days by reverse transcription-polymerase chain reaction. Results: Although diabetic ADSCs demonstrated decreased responses compared to normal ADSCs, dHACM treatment stimulated diabetic ADSCs to proliferate after 3 days and enhanced migration over 24 h, similar to normal ADSCs. dHACM-treated diabetic ADSCs modulated secretion of soluble signals, including regulators of inflammation, angiogenesis, and healing. All ADSCs evaluated also responded to dHACM treatment with altered expression of immunomodulatory genes, including interleukins (IL)-1α, IL-1ß, and IL-1RA. Innovation: This is the first reported case demonstrating that diabetic ADSCs respond to novel amniotic membrane therapies, specifically treatment with dHACM. Conclusion: dHACM stimulated diabetic ADSCs to migrate, proliferate, and alter cytokine expression suggesting that, despite their diabetic origin, ADSCs may respond to dHACM to accelerate diabetic wound healing.

Dehydrated human amnion/chorion membrane regulates stem cell activity in vitro.

Human-derived placental tissues have been shown in randomized clinical trials to be effective for healing chronic wounds, and have also demonstrated the ability to recruit stem cells to the wound site in vitro and in vivo. In this study, PURION(®) Processed dehydrated human amnion/chorion membrane allografts (dHACM, EpiFix(®) , MiMedx Group, Marietta, GA) were evaluated for their ability to alter stem cell activity in vitro. Human bone marrow mesenchymal stem cells (BM-MSCs), adipose derived stem cells (ADSCs), and hematopoietic stem cells (HSCs) were treated with soluble extracts of dHACM tissue, and were evaluated for cellular proliferation, migration, and cytokine secretion. Stem cells were analyzed for cell number by DNA assay after 24 h, closure of an acellular zone using microscopy over 3 days, and soluble cytokine production in the medium of treated stem cells was analyzed after 3 days using a multiplex ELISA array. Treatment with soluble extracts of dHACM tissue stimulated BM-MSCs, ADSCs, and HSCs to proliferate with a significant increase in cell number after 24 h. dHACM treatment accelerated closure of an acellular zone by ADSCs and BM-MSCs after 3 days, compared to basal medium. BM-MSCs, ADSCs, and HSCs also modulated endogenous production of a number of various soluble signals, including regulators of inflammation, mitogenesis, and wound healing. dHACM treatment promoted increased proliferation and migration of ADSCs, BM-MSCs, and HSCs, along with modulation of secreted proteins from those cells. Therefore, dHACM may impact wound healing by amplifying host stem cell populations and modulating their responses in treated wound tissues. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 104B: 1495-1503, 2016.

Percutaneous injection of augment injectable bone graft (rhPDGF-BB and ß-tricalcium phosphate [ß-TCP]/bovine type I collagen matrix) increases vertebral bone mineral density in geriatric female baboons.

BACKGROUND CONTEXT: Recombinant human platelet-derived growth factor-BB (rhPDGF-BB) homodimer is a chemotactic, mitogenic, and angiogenic factor expressed by platelets. This biological triad is profoundly important in the bone regenerative cascade. Therefore, the expectation was that rhPDGF-BB locally administered to designated lumbar vertebrae in a soluble Type I bovine collagen/ß-tricalcium phosphate (ß-TCP) injectable paste would have an osteoanabolic effect. PURPOSE: The study objective focused on safety and efficacy of the rhPDGF-BB and soluble Type I bovine collagen/ß-TCP to increase bone density when injected directly into specific lumbar vertebral bodies in elderly (17- to 18-year-old) female baboons. STUDY DESIGN/SETTING: The study was designed to determine whether vertebral bone mineral density (BMD) in aged female baboons could be increased by locally administering recombinant rhPDGF-BB combined in a soluble Type I bovine collagen/ß-TCP paste formulation. METHODS: A total of six baboons were divided equally into two groups. Group 1 received 1.0 mg/mL rhPDGF-BB in 20 mM sodium acetate plus soluble Type I bovine collagen/ß-TCP. Group 2 was treated with 20 mM sodium acetate plus soluble Type I bovine collagen/ß-TCP. Baboons in each group also received a sham surgery. Surgery was conducted using a percutaneous, fluoroscopically guided approach, and quantitative computed tomography (qCT) and radiographs were done at dedicated time periods. The qCT was used to determine volumetric BMD (vBMD). At euthanasia (36-week posttreatment), lumbar vertebrae were recovered and analyzed by qCT scans and histology. Funds were received to support this work from BioMimetic Therapeutics, Inc. The device that is the subject of this manuscript is not Food Drug Administration approved for this indication and is not commercially available in the United States. RESULTS: The qCT and histopathological data suggested that vBMD and bone morphology increased significantly in the lumbar vertebrae treated with the rhPDGF-BB-containing composition. CONCLUSIONS: Bone mineral density and bone morphology quality of lumbar vertebrae in aged female baboons were improved by direct injection of rhPDGF-BB in a soluble Type I bovine collagen/ß-TCP paste. Throughout the course of the study, there were neither local nor systemic adverse effects.

Preclinical Toxicology Studies of Recombinant Human Platelet-Derived Growth Factor-BB Either Alone or in Combination with Beta-Tricalcium Phosphate and Type I Collagen.

Human platelet-derived growth factor-BB (hPDGF-BB) is a basic polypeptide growth factor released from platelets at the injury site. It is a multifunctional molecule that regulates DNA synthesis and cell division and induces biological effects that are implicated in tissue repair, atherosclerosis, inflammatory responses, and neoplastic diseases. This paper is an overview of the toxicology data generated from a broad testing platform to determine bone, soft tissue, and systemic responses following administration of rhPDGF-BB. Moreover, the systemic and local toxicity of recombinant human PDGF-BB (rhPDGF-BB) in combination with either beta-tricalcium phosphate (ß-TCP) or collagen combined with ß-TCP was studied to determine dermal sensitization, irritation, intramuscular tissue responses, pyrogenicity, genotoxicity, and hemolytic properties. All data strongly suggest that rhPDGF-BB either alone or in combination with ß-TCP or collagen with ß-TCP is biocompatible and has neither systemic nor local toxicity, supporting its safe use in enhancing wound healing in patients.

Reconstructing mandibular defects using autologous tissue-engineered tooth and bone constructs.

PURPOSE: Current strategies for jaw reconstruction require multiple operations to replace bone and teeth. To improve on these methods, we investigated simultaneous mandibular and tooth reconstruction, using a Yucatan minipig model. MATERIALS AND METHODS: Tooth and bone constructs were prepared from third molar tooth tissue and iliac-crest bone marrow-derived osteoblasts isolated from, and implanted back into, the same pig as an autologous reconstruction. Implants were harvested after 12 and 20 weeks and evaluated by x-ray, ultrahigh-resolution volume computed tomographic (VCT), histological, and immunohistochemical analyses. RESULTS: Small tooth structures were identified, and consisted of organized dentin, enamel, pulp, and periodontal ligament tissues, surrounded by new bone. No dental tissues formed in implants without tooth-bud cells, and bone regeneration was observed to a limited extent. Immunohistochemical analyses using tooth-specific and bone-specific antibodies confirmed the identity of regenerated tissues. CONCLUSIONS: This pilot study supports the feasibility of tissue-engineering approaches for coordinated autologous tooth and mandible reconstruction, and provides a basis for future improvement of this technique for eventual clinical use in humans.

Tissue-engineered hybrid tooth and bone.

Tooth loss accompanied by alveolar bone resorption presents a significant clinical problem. We have investigated the utility of a tissue-engineering approach to provide corrective therapies for tooth-bone loss. Hybrid tooth-bone tissues were bioengineered as follows. Tooth implants were generated from pig third molar tooth bud cells seeded onto polyglycolide (PGA) and polyglycolide-colactide (PLGA) scaffolds, and grown for 4 weeks in the omenta of adult rat hosts. Bone implants were generated from osteoblasts induced from bone marrow progenitor cells obtained from the same pig, seeded onto PLGA fused wafer scaffolds, and grown for 10 days in a rotational oxygen-permeable bioreactor system. The tooth and bone implants were harvested, sutured together, reimplanted, and grown in the omenta for an additional 8 weeks. Histological and immunohistochemical analyses of the excised hybrid tooth-bone constructs revealed the presence of tooth tissues, including primary and reparative dentin and enamel in the tooth portion of hybrid tooth-bone implants, and osteocalcin and bone sialoprotein-positive bone in the bone portion of hybrid tooth-bone constructs. Collagen type III-positive connective tissue resembling periodontal ligament and tooth root structures were present at the interface of bioengineered tooth and bone tissues. These results demonstrate the utility of a hybrid tooth-bone tissue-engineering approach for the eventual clinical treatment of tooth loss accompanied by alveolar bone resorption.

Fluoride induces endoplasmic reticulum stress in ameloblasts responsible for dental enamel formation.

The mechanism of how fluoride causes fluorosis remains unknown. Exposure to fluoride can inhibit protein synthesis, and this may also occur by agents that cause endoplasmic reticulum (ER) stress. When translated proteins fail to fold properly or become misfolded, ER stress response genes are induced that together comprise the unfolded protein response. Because ameloblasts are responsible for dental enamel formation, we used an ameloblast-derived cell line (LS8) to characterize specific responses to fluoride treatment. LS8 cells were growth-inhibited by as little as 1.9-3.8 ppm fluoride, whereas higher doses induced ER stress and caspase-mediated DNA fragmentation. Growth arrest and DNA damage-inducible proteins (GADD153/CHOP, GADD45alpha), binding protein (BiP/glucose-responsive protein 78 (GRP78), the non-secreted form of carbonic anhydrase VI (CA-VI), and active X-box-binding protein-1 (Xbp-1) were all induced significantly after exposure to 38 ppm fluoride. Unexpectedly, DNA fragmentation increased when GADD153 expression was inhibited by short interfering RNA treatment but remained unaffected by transient GADD153 overexpression. Analysis of control and GADD153(-/-) embryonic fibroblasts demonstrated that caspase-3 mediated the increased DNA fragmentation observed in the GADD153 null cells. We also demonstrate that mouse incisor ameloblasts are sensitive to the toxic effects of high dose fluoride in drinking water. Activated Ire1 initiates an ER stress response pathway, and mouse ameloblasts were shown to express activated Ire1. Ire1 levels appeared induced by fluoride treatment, indicating that ER stress may play a role in dental fluorosis. Low dose fluoride, such as that present in fluoridated drinking water, did not induce ER stress.