Changes in oral health during aging in a novel non-human primate model.

Oral health plays a significant role in the quality of life and overall well-being of the aging population. However, age-related changes in oral health are not well understood due to challenges with current animal models. In this study, we analyzed the oral health and microbiota of a short-lived non-human primate (i.e., marmoset), as a step towards establishing a surrogate for studying the changes that occur in oral health during human aging. We investigated the oral health of marmosets using cadaveric tissues in three different cohorts: young (aged ≤6 years), middle-aged, and older (>10 years) and assessed the gingival bacterial community using analyses of the V3-V4 variable region of 16S rRNA gene. The oldest cohort had a significantly higher number of dental caries, increased dental attrition/erosion, and deeper periodontal pocket depth scores. Oral microbiome analyses showed that older marmosets had a significantly greater abundance of Escherichia-Shigella and Propionibacterium, and a lower abundance of Agrobacterium/Rhizobium at the genus level. Alpha diversity of the microbiome between the three groups showed no significant differences; however, principal coordinate analysis and non-metric multidimensional scaling analysis revealed that samples from middle-aged and older marmosets were more closely clustered than the youngest cohort. In addition, linear discriminant analysis effect size (LEFSe) identified a higher abundance of Esherichia-Shigella as a potential pathogenic biomarker in older animals. Our findings confirm that changes in the oral microbiome are associated with a decline in oral health in aging marmosets. The current study suggests that the marmoset model recapitulates some of the changes in oral health associated with human aging and may provide opportunities for developing new preventive strategies or interventions which target these disease conditions.

Extracellular matrix turnover in salivary gland disorders and regenerative therapies: Obstacles and opportunities.

Salivary gland (SG) extracellular matrix (ECM) has a major influence on tissue development, homeostasis, and tissue regeneration after injury. During aging, disease, and physical insult, normal remodeling of the SG microenvironment (i.e. ECM) becomes dysregulated, leading to alterations in matrix composition which disrupt tissue architecture/structure, alter cell activity, and negatively impact gland function. Matrix metalloproteinases (MMPs) are a large and diverse family of metalloendopeptidases which play a major role in matrix degradation and are intimately involved in regulating development and cell function; dysregulation of these enzymes leads to the production of a fibrotic matrix. In the SG this altered fibrotic ECM (or cell microenvironment) negatively impacts normal cell function and the effectiveness of gene and stem cell therapies which serve as a foundation for many SG regenerative therapies. For this reason, prospective regenerative strategies should prioritize the maintenance and/or restoration of a healthy SG ECM. Mesenchymal stem cells (MSCs) have great potential for mitigating damage to the SG microenvironment by ameliorating inflammation, reducing fibrosis, and repairing the damaged milieu of extracellular regulatory cues, including the matrix. This review addresses our current understanding of the impact of aging and disease on the SG microenvironment and suggests critical deficiencies and opportunities in ECM-targeted therapeutic interventions.

Autologous mesenchymal stem cells offer a new paradigm for salivary gland regeneration.

Salivary gland (SG) dysfunction, due to radiotherapy, disease, or aging, is a clinical manifestation that has the potential to cause severe oral and/or systemic diseases and compromise quality of life. Currently, the standard-of-care for this condition remains palliative. A variety of approaches have been employed to restore saliva production, but they have largely failed due to damage to both secretory cells and the extracellular matrix (niche). Transplantation of allogeneic cells from healthy donors has been suggested as a potential solution, but no definitive population of SG stem cells, capable of regenerating the gland, has been identified. Alternatively, mesenchymal stem cells (MSCs) are abundant, well characterized, and during SG development/homeostasis engage in signaling crosstalk with the SG epithelium. Further, the trans-differentiation potential of these cells and their ability to regenerate SG tissues have been demonstrated. However, recent findings suggest that the "immuno-privileged" status of allogeneic adult MSCs may not reflect their status post-transplantation. In contrast, autologous MSCs can be recovered from healthy tissues and do not present a challenge to the recipient's immune system. With recent advances in our ability to expand MSCs in vitro on tissue-specific matrices, autologous MSCs may offer a new therapeutic paradigm for restoration of SG function.

Organ-specific extracellular matrix directs trans-differentiation of mesenchymal stem cells and formation of salivary gland-like organoids in vivo.

BACKGROUND: Current treatments for salivary gland (SG) hypofunction are palliative and do not address the underlying cause or progression of the disease. SG-derived stem cells have the potential to treat SG hypofunction, but their isolation is challenging, especially when the tissue has been damaged by disease or irradiation for head and neck cancer. In the current study, we test the hypothesis that multipotent bone marrow-derived mesenchymal stem cells (BM-MSCs) in a rat model are capable of trans-differentiating to the SG epithelial cell lineage when induced by a native SG-specific extracellular matrix (SG-ECM) and thus may be a viable substitute for repairing damaged SGs. METHODS: Rat BM-MSCs were treated with homogenates of decellularized rat SG-ECM for one hour in cell suspension and then cultured in tissue culture plates for 7 days in growth media. By day 7, the cultures contained cell aggregates and a cell monolayer. The cell aggregates were hand-selected under a dissecting microscope, transferred to a new tissue culture dish, and cultured for an additional 7 days in epithelial cell differentiation media. Cell aggregates and cells isolated from the monolayer were evaluated for expression of SG progenitor and epithelial cell specific markers, cell morphology and ultrastructure, and ability to form SG-like organoids in vivo. RESULTS: The results showed that this approach was very effective and guided the trans-differentiation of a subpopulation of CD133-positive BM-MSCs to the SG epithelial cell lineage. These cells expressed amylase, tight junction proteins (Cldn 3 and 10), and markers for SG acinar (Aqp5 and Mist 1) and ductal (Krt 14) cells at both the transcript and protein levels, produced intracellular secretory granules which were morphologically identical to those found in submandibular gland, and formed SG-like organoids when implanted in the renal capsule in vivo. CONCLUSIONS: The results of this study suggest the feasibility of using autologous BM-MSCs as an abundant source of stem cells for treating SG hypofunction and restoring the production of saliva in these patients.

Matrix-bound Cyr61/CCN1 is required to retain the properties of the bone marrow mesenchymal stem cell niche but is depleted with aging.

Previously, we showed that extracellular matrices (ECMs), produced ex vivo by various types of stromal cells, direct bone marrow mesenchymal stem cells (BM-MSCs) in a tissue-specific manner and recapitulate physiologic changes characteristic of the aging microenvironment. In particular, BM-MSCs obtained from elderly donors and cultured on ECM produced by young BM stromal cells showed improved quantity, quality and osteogenic differentiation. In the present study, we searched for matrix components that are required for a functional BM-MSC niche by comparing ECMs produced by BM stromal cells from "young" (≤25 y/o) versus "elderly" (≥60 y/o) donors. With increasing donor age, ECM fibrillar organization and mechanical integrity deteriorated, along with the ability to promote BM-MSC proliferation and responsiveness to growth factors. Proteomic analyses revealed that the matricellular protein, Cyr61/CCN1, was present in young, but undetectable in elderly, BM-ECM. To assess the role of Cyr61 in the BM-MSC niche, we used genetic methods to down-regulate the incorporation of Cyr61 during production of young ECM and up-regulate its incorporation in elderly ECM. The results showed that Cyr61-depleted young ECM lost the ability to promote BM-MSC proliferation and growth factor responsiveness. However, up-regulating the incorporation of Cyr61 during synthesis of elderly ECM restored its ability to support BM-MSC responsiveness to osteogenic factors such as BMP-2 and IGF-1. We next examined aging bone and compared bone mineral density and Cyr61 content of L4-L5 vertebral bodies in "young" (9-11 m/o) and "elderly" (21-33 m/o) mice. Our analyses showed that low bone mineral density was associated with decreased amounts of Cyr61 in osseous tissue of elderly versus young mice. Our results strongly demonstrate a novel role for ECM-bound Cyr61 in the BM-MSC niche, where it is responsible for retention of BM-MSC proliferation and growth factor responsiveness, while depletion of Cyr61 from the BM niche contributes to an aging-related dysregulation of BM-MSCs. Our results also suggest new potential therapeutic targets for treating age-related bone loss by restoring specific ECM components to the stem cell niche.

Culture on a native bone marrow-derived extracellular matrix restores the pancreatic islet basement membrane, preserves islet function, and attenuates islet immunogenicity.

Islet transplantation in man is limited by multiple factors including islet availability, islet cell damage caused by collagenase during isolation, maintenance of islet function between isolation and transplantation, and allograft rejection. In this study, we describe a new approach for preparing islets that enhances islet function in vitro and reduces immunogenicity. The approach involves culture on native decellularized 3D bone marrow-derived extracellular matrix (3D-ECM), which contains many of the matrix components present in pancreas, prior to islet transplantation. Compared to islets cultured on tissue culture plastic (TCP), islets cultured on 3D-ECM exhibited greater attachment, higher survival rate, increased insulin content, and enhanced glucose-stimulated insulin secretion. In addition, culture of islets on 3D-ECM promoted recovery of vascular endothelial cells within the islets and restored basement membrane-related proteins (eg, fibronectin and collagen type VI). More interestingly, culture on 3D-ECM also selectively decontaminated islets of "passenger" cells (co-isolated with the islets) and restored basement membrane-associated type VI collagen, which were associated with an attenuation in islet immunogenicity. These results demonstrate that this novel approach has promise for overcoming two major issues in human islet transplantation: (a) poor yield of islets from donated pancreas tissue and (b) the need for life-long immunosuppression.

Oral and Craniofacial Stem Cells: An Untapped Source for Neural Tissue Regeneration.

Nerve tissue regeneration continues to represent an intractable obstacle to realizing the promise of tissue engineering. Although neurobiology works to shed light on the mechanisms governing neuronal growth and repair, considerable technical gaps remain that hinder progress. Chief among these is the absence of an appropriate culture environment to faithfully reproduce the neuronal niche ex vivo. We propose that the various multipotent cells found in the oral cavity may represent an important yet underutilized resource for preparing such neurogenic microenvironments. Similar to those of nerve tissue, these cell populations are of ectodermal origin and have clinically demonstrated neurogenic potential. Although there is a lack of consensus on whether putative types of oral and craniofacial stem cells constitute distinct populations, their contribution to neural tissue engineering may be twofold: as a cellular feedstock for neoneurogenesis and for the production of specialized in vitro environments for neurogenic differentiation, phenotype maintenance, and use in therapeutic applications. Impact statement We propose that addressing gaps in understanding the neurogenic role of dental stem cells and their microenvironment may yield efficient and reliable strategies for long-term neuronal cell culture and open new avenues for neural regeneration in both dental, nerve, and other tissues.

Native extracellular matrix, synthesized ex vivo by bone marrow or adipose stromal cells, faithfully directs mesenchymal stem cell differentiation.

Mesenchymal stem cells (MSCs) are highly responsive to cues in the microenvironment (niche) that must be recapitulated ex vivo to study their authentic behavior. In this study, we hypothesized that native bone marrow (BM)- and adipose (AD)-derived extracellular matrices (ECM) were unique in their ability to control MSC behavior. To test this, we compared proliferation and differentiation of bone marrow (BM)-derived MSCs when maintained on native decellularized ECM produced by BM versus AD stromal cells (i.e. BM- versus AD-ECM). We found that both ECMs contained similar types of collagens but differed in the relative abundance of each. Type VI collagen was the most abundant (≈60% of the total collagen present), while type I was the next most abundant at ≈30%. These two types of collagen were found in nearly equal proportions in both ECMs. In contrast, type XII collagen was almost exclusively found in AD-ECM, while types IV and V were only found in BM-ECM. Physically and mechanically, BM-ECM was rougher and stiffer, but less adhesive, than AD-ECM. During 14 days in culture, both ECMs supported BM-MSC proliferation better than tissue culture plastic (TCP), although MSC-related surface marker expression remained relatively high on all three culture surfaces. BM-MSCs cultured in osteogenic (OS) differentiation media on BM-ECM displayed a significant increase in calcium deposition in the matrix, indicative of osteogenesis, while BM-MSCs cultured on AD-ECM in the presence of adipogenic (AP) differentiation media showed a significant increase in Oil Red O staining, indicative of adipogenesis. Further, culture on BM-ECM significantly increased BM-MSC-responsiveness to rhBMP-2 (an osteogenic inducer), while culture on AD-ECM enhanced responsiveness to rosiglitazone (an adipogenic inducer). These findings support our hypothesis and indicate that BM- and AD-ECMs retain unique elements, characteristic of their tissue-specific microenvironment (niche), which promote retention of MSC differentiation state (i.e. "stemness") during expansion and direct cell response to lineage-specific inducers. This study provides a new paradigm for precisely controlling MSC fate to a desired cell lineage for tissue-specific cell-based therapies.

In vivo hydroxyapatite scaffold performance in infected bone defects.

Critically sized bone defects are often compounded by infectious complications. The standard of care consists of bone autografts with systemic antibiotics. These injuries and treatments lead to donor site morbidity, antibiotic resistant strains of bacteria, and often end stage amputation. This study proposes an alternative to the autograft using a porous, hydroxyapatite (HA) scaffold evaluated with and without infection and antibiotics. Twenty-four New Zealand white rabbits received either our HA scaffold or a pulverized autograft (PBA) within a surgically created critical-sized defect in the femur. The two grafts were evaluated in either septic or aseptic defects and with or without antibiotic treatment. The HA scaffolds were characterized with micro computed tomography. Post-euthanasia, micro computed tomography, histology, and white blood cells component analysis were completed. The HA had significantly greater (p < .001) mineralization to total volume than the PBA groups with 27.56% and 14.88%, respectively, and the septic HA groups were significantly greater than the aseptic groups both with and without antibiotics (p = .016). The bone quality denoted by bone mineral density was also significantly greater (p < .001) in the HA groups (67.01 ± 0.38 mgHA/cm3 ) than the PBA groups (64.66 ± 0.85 mgHA/cm3 ). The HA scaffold is a viable alternative to the bone autograft in defects with and without infection as shown by the quality and quantity of bone.

Restoring the quantity and quality of elderly human mesenchymal stem cells for autologous cell-based therapies.

BACKGROUND: Degenerative diseases are a major public health concern for the aging population and mesenchymal stem cells (MSCs) have great potential for treating many of these diseases. However, the quantity and quality of MSCs declines with aging, limiting the potential efficacy of autologous MSCs for treating the elderly population. METHODS: Human bone marrow (BM)-derived MSCs from young and elderly donors were obtained and characterized using standard cell surface marker criteria (CD73, CD90, CD105) as recommended by the International Society for Cellular Therapy (ISCT). The elderly MSC population was isolated into four subpopulations based on size and stage-specific embryonic antigen-4 (SSEA-4) expression using fluorescence-activated cell sorting (FACS), and subpopulations were compared to the unfractionated young and elderly MSCs using assays that evaluate MSC proliferation, quality, morphology, intracellular reactive oxygen species, ß-galactosidase expression, and adenosine triphosphate (ATP) content. RESULTS: The ISCT-recommended cell surface markers failed to detect any differences between young and elderly MSCs. Here, we report that elderly MSCs were larger in size and displayed substantially higher concentrations of intracellular reactive oxygen species and ß-galactosidase expression and lower amounts of ATP and SSEA-4 expression. Based on these findings, cell size and SSEA-4 expression were used to separate the elderly MSCs into four subpopulations by FACS. The original populations (young and elderly MSCs), as well as the four subpopulations, were then characterized before and after culture on tissue culture plastic and BM-derived extracellular matrix (BM-ECM). The small SSEA-4-positive subpopulation representing ~ 8% of the original elderly MSC population exhibited a "youthful" phenotype that was similar to that of young MSCs. The biological activity of this elderly subpopulation was inhibited by senescence-associated factors produced by the unfractionated parent population. After these "youthful" cells were isolated and expanded (three passages) on a "young microenvironment" (i.e., BM-ECM produced by BM cells from young donors), the number of cells increased ≈ 17,000-fold to 3 × 109 cells and retained their "youthful" phenotype. CONCLUSIONS: These results suggest that it is feasible to obtain large numbers of high-quality autologous MSCs from the elderly population and establish personal stem cell banks that will allow serial infusions of "rejuvenated" MSCs for treating age-related diseases.

Umbilical cord blood-derived non-hematopoietic stem cells retrieved and expanded on bone marrow-derived extracellular matrix display pluripotent characteristics.

BACKGROUND: Umbilical cord blood (UCB) not only contains hematopoietic stem cells (HSCs), but also non-hematopoietic stem cells (NHSCs) that are able to differentiate into a number of distinct cell types. Based on studies published to date, the frequency of NHSCs in UCB is believed to be very low. However, the isolation of these cells is primarily based on their adhesion to tissue culture plastic surfaces. METHODS AND RESULTS: In the current study, we demonstrate that this approach overlooks some of the extremely immature NHSCs because they lack the ability to adhere to plastic. Using a native extracellular matrix (ECM), produced by bone marrow (BM) stromal cells, the majority of the UCB-NHSCs attached within 4 h. The colony-forming unit fibroblast frequency of these cells was 1.5 × 104/108 mononuclear cells, which is at least 4000-fold greater than previously reported for UCB-NHSCs. The phenotype of these cells was fibroblast-like and different from those obtained by plastic adhesion; they formed embryonic body-like clusters that were OCT4-positive and expressed other human embryonic stem cell-related markers. Importantly, when implanted subcutaneously for 8 weeks into immunocompromised mice, these ECM-adherent and expanded NHSCs generated three germ layer-derived human tissues including muscle, fat, blood vessel, bone, gland, and nerve. Moreover, injection of these cells into muscle damaged by cryoinjury significantly accelerated muscle regeneration. CONCLUSIONS: These results indicate that UCB may be a virtually unlimited source of NHSCs when combined with isolation and expansion on ECM. NHSCs may be a practical alternative to embryonic stem cells for a number of therapeutic applications.

One size does not fit all: developing a cell-specific niche for in vitro study of cell behavior.

For more than 100years, cells and tissues have been studied in vitro using glass and plastic surfaces. Over the last 10-20years, a great body of research has shown that cells are acutely sensitive to their local environment (extracellular matrix, ECM) which contains both chemical and physical cues that influence cell behavior. These observations suggest that modern cell culture systems, using tissue culture polystyrene (TCP) surfaces, may fail to reproduce authentic cell behavior in vitro, resulting in "artificial outcomes." In the current study, we use bone marrow (BM)- and adipose (AD)-derived stromal cells to prepare BM-ECM and AD-ECM, which are decellularized after synthesis by the cells, to mimic the cellular niche for each of these tissues. Each ECM was characterized for its ability to affect BM- and AD-mesenchymal stem cell (MSC) proliferation, as well as proliferation of three cancer cell lines (HeLa, MCF-7, and MDA-MB-231), modulate cell spreading, and direct differentiation relative to standard TCP surfaces. We found that both ECMs promoted the proliferation of MSCs, but that this effect was enhanced when the tissue-origin of the cells matched that of the ECM (i.e. BM-ECM promoted the proliferation of BM-MSCs over AD-MSCs, and vice versa). Moreover, BM- and AD-ECM were shown to preferentially direct MSC differentiation towards either osteogenic or adipogenic lineage, respectively, suggesting that the effects of the ECM were tissue-specific. Further, each ECM influenced cell morphology (i.e. circularity), irrespective of the origin of the MSCs, lending more support to the idea that effects were tissue specific. Interestingly, unlike MSCs, these ECMs did not promote the proliferation of the cancer cells. In an effort to further understand how these three culture substrates influence cell behavior, we evaluated the chemical (protein composition) and physical properties (architecture and mechanical) of the two ECMs. While many structural proteins (e.g. collagen and fibronectin) were found at equivalent levels in both BM- and AD-ECM, the architecture (i.e. fiber orientation; surface roughness) and physical properties (storage modulus, surface energy) of each were unique. These results, demonstrating differences in cell behavior when cultured on the three different substrates (BM- and AD-ECM and TCP) with differences in chemical and physical properties, provide evidence that the two ECMs may recapitulate specific elements of the native stem cell niche for bone marrow and adipose tissues. More broadly, it could be argued that ECMs, elaborated by cells ex vivo, serve as an ideal starting point for developing tissue-specific culture environments. In contrast to TCP, which relies on the "one size fits all" paradigm, native tissue-specific ECM may be a more rational model to approach engineering 3D tissue-specific culture systems to replicate the in vivo niche. We suggest that this approach will provide more meaningful information for basic research studies of cell behavior as well as cell-based therapeutics.

Native extracellular matrix preserves mesenchymal stem cell "stemness" and differentiation potential under serum-free culture conditions.

INTRODUCTION: Bone marrow-derived mesenchymal stem cells (BM-MSCs) for clinical use should not be grown in media containing fetal bovine serum (FBS), because of serum-related concerns over biosafety and batch-to-batch variability. Previously, we described the preparation and use of a cell-free native extracellular matrix (ECM) made by bone marrow cells (BM-ECM) which preserves stem cell properties and enhances proliferation. Here, we compare colony-forming ability and differentiation of MSCs cultured on BM-ECM with a commercially available matrix (CELLstart™) and tissue culture plastic (TCP) under serum-free conditions. METHODS: Primary MSCs from freshly isolated bone marrow-derived mononuclear cells or passaged MSCs (P1) were grown in serum-containing (SCM) or serum-free (SFM) media on BM-ECM, CELLstart™, or TCP substrates. Proliferation, cell composition (phenotype), colony-forming unit replication, and bone morphogenetic protein-2 (BMP-2) responsiveness were compared among cells maintained on the three substrates. RESULTS: Proliferation of primary BM-MSCs was significantly higher in SCM than SFM, irrespectively of culture substrate, suggesting that the expansion of these cells requires SCM. In contrast, passaged cells cultured on BM-ECM or CELLstart™ in SFM proliferated to nearly the same extent as cells in SCM. However, morphologically, those on BM-ECM were smaller and more aligned, slender, and long. Cells grown for 7 days on BM-ECM in SFM were 20-40 % more positive for MSC surface markers than cells cultured on CELLstart™. Cells cultured on TCP contained the smallest number of cells positive for MSC markers. MSC colony-forming ability in SFM, as measured by CFU-fibroblasts, was increased 10-, 9-, and 2-fold when P1 cells were cultured on BM-ECM, CELLstart™, and TCP, respectively. Significantly, CFU-adipocyte and -osteoblast replication of cells grown on BM-ECM was dramatically increased over those on CELLstart™ (2X) and TCP (4-7X). BM-MSCs, cultured in SFM and treated with BMP-2, retained their differentiation capacity better on BM-ECM than on either of the other two substrates. CONCLUSIONS: Our findings indicate that BM-ECM provides a unique microenvironment that supports the colony-forming ability of MSCs in SFM and preserves their stem cell properties. The establishment of a robust culture system, combining native tissue-specific ECM and SFM, provides an avenue for preparing significant numbers of potent MSCs for cell-based therapies in patients.

Secretion of salivary statherin is compromised in uncontrolled diabetic patients.

BACKGROUND: Statherin is an important salivary protein for maintaining oral health. The purpose of the current study was to determine if differences in statherin levels exist between diabetic and healthy subjects. METHODS: A total of 48 diabetic and healthy controls were randomly selected from a community-based database. Diabetic subjects (n=24) had fasting glucose levels >180 mg/dL, while controls (n=24) had levels <110 mg/dL. Parotid saliva (PS) and sublingual/submandibular saliva (SS) were collected and salivary flow rates determined. Salivary statherin levels were determined by densitometry of Western blots. Blood hemoglobin A1c (HbA1c) and total protein in saliva were also obtained. RESULTS: SS, but not PS, salivary flow rate and total protein in diabetics were significantly less than in healthy controls (p=0.021 & p<0.001 respectively). Correlation analysis revealed the existence of a negative correlation between PS statherin levels and HbA1c (p=0.012) and fasting glucose (p=0.021) levels, while no such correlation was found for SS statherin levels. When statherin levels were normalized to total salivary protein, the proportion of PS statherin, but not SS statherin, in diabetics was significantly less than controls (p=0.032). In contrast, the amount of statherin secretion in SS, but not PS, was significantly decreased in diabetics compared to controls (p=0.016). CONCLUSIONS AND GENERAL SIGNIFICANCE: The results show that synthesis and secretion of statherin is reduced in diabetics and this reduction is salivary gland specific. As compromised salivary statherin secretion leads to increased oral health risk, this study indicates that routine oral health assessment of these patients is warranted.

Silk fibroin scaffolds promote formation of the ex vivo niche for salivary gland epithelial cell growth, matrix formation, and retention of differentiated function.

Salivary gland hypofunction often results from a number of causes, including the use of various medications, radiation for head and neck tumors, autoimmune diseases, diabetes, and aging. Since treatments for this condition are lacking and adult salivary glands have little regenerative capacity, there is a need for cell-based therapies to restore salivary gland function. Development of these treatment strategies requires the establishment of a system that is capable of replicating the salivary gland cell "niche" to support the proliferation and differentiation of salivary gland progenitor cells. In this study, a culture system using three-dimensional silk fibroin scaffolds (SFS) and primary salivary gland epithelial cells (pSGECs) from rat submandibular (SM) gland and parotid gland (PG) was established and characterized. pSGECs grown on SFS, but not tissue culture plastic (TCP), formed aggregates of cells with morphological features resembling secretory acini. High levels of amylase were released into the media by both cell types after extended periods in culture on SFS. Remarkably, cultures of PG-derived cells on SFS, but not SM cells, responded to isoproterenol, a ß-adrenergic receptor agonist, with increased enzyme release. This behavior mimics that of the salivary glands in vivo. Decellularized extracellular matrix (ECM) formed by pSGECs in culture on SFS contained type IV collagen, a major component of the basement membrane. These results demonstrate that pSGECs grown on SFS, but not TCP, retain important functional and structural features of differentiated salivary glands and produce an ECM that mimics the native salivary gland cell niche. These results demonstrate that SFS has potential as a scaffold for creating the salivary gland cell niche in vitro and may provide an approach for inducing multipotent stem cells to provide therapeutically meaningful numbers of salivary gland progenitor cells for regenerating these tissues in patients.

In vivo performance of combinations of autograft, demineralized bone matrix, and tricalcium phosphate in a rabbit femoral defect model.

Large bone defects may be treated with autologous or allogeneic bone preparations. Each treatment has advantages and disadvantages; therefore, a clinically viable option for treating large (e.g., gap) bone defects may be a combination of the two. In the present study, bone repair was determined with combinations of autografts, allografts, and synthetic bone grafts using an established rabbit femoral defect model. Bilateral unicortical femoral defects were surgically prepared and treated with combinatorial bone grafts according to one of seven treatment groups. Recipient sites were retrieved at six weeks. Cellular/tissue responses and new bone formation were assessed by histology and histomorphometry. Histological analysis images indicated neither evidence of inflammatory, immune responses, tissue necrosis, nor osteolysis. Data suggested co-integration of implanted agents with host and newly formed bone. Finally, the histomorphometric data suggested that the tricalcium phosphate-based synthetic bone graft substitute allowed new bone formation that was similar to the allograft (i.e., demineralized bone matrix, DBM).

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.

Evaluation of BMP-2 tethered polyelectrolyte coatings on hydroxyapatite scaffolds in vivo.

The goal of this in vivo study was to evaluate the osteoinductive and angio-inductive properties of a porous hydroxyapatite (HAp) scaffold with immobilized recombinant bone morphogenetic protein-2 (rhBMP-2) on the surface. It was hypothesized in this study that the use of a rhBMP-2 incorporated polyelectrolyte coating on the HAp scaffold would allow for controlled exposure of rhBMP-2 into the tissue and would provide a sound platform for tissue growth. The scaffolds were characterized for porosity and interconnectivity using pycnometry, scanning electron microscopy and micro-ct. These scaffolds were then divided into the following four groups: (a) HAp scaffold (n-HAp group), (b) rhBMP-2 physically adsorbed on HAp scaffold (HAp-BMP-2 Group), (c) polyelectrolyte coating on HAp scaffold without rhBMP-2 (HAp-PEI Scaffold Group), and (d) polyelectrolyte coating tethered with rhBMP-2 on HAp scaffold (HAp-PEI-BMP-2 Scaffold Group). Using 18 skeletally matured New Zealand white rabbits, these scaffolds were evaluated in a nonload bearing femoral condyle plug model. The negative controls for this study have defects that were left untreated and the positive controls have defects that were filled with autologous bone graft harvested from epsilateral iliac crest. Bone induction, vessel growth, and scaffold-bone contact were analyzed after 8-week implantation using micro-CT and histomorphometry. It was concluded from this study that the use of scaffold with an attached rhBMP-2 increased the vascularization around the implant when compared with the uncoated n-HAp scaffold, a necessary step of bone regeneration. The open-pore HAp scaffold was also concluded to provide a platform for tissue growth, drug loading, and tissue interaction.

Rapid-prototyped PLGA/ß-TCP/hydroxyapatite nanocomposite scaffolds in a rabbit femoral defect model.

Bone tissue engineering scaffolds composed of poly(d,l-lactide:glycolide) (DL-PLGA) and ß-tricalcium phosphate (ß-TCP) nanocomposites were prepared and characterized. Scaffolds with two specific architectures were produced via fused deposition modeling (FDM), a type of extrusion freeform fabrication. Microfilaments deposited at angles of 0° and 90° were designated as the 'simple' scaffold architecture, while those deposited at angles alternating between 0°, 90°, 45° and -45° were designated as the 'complex' scaffold architecture. In addition, the simple and complex scaffolds were coated with hydroxyapatite (HA). The surface morphology of the scaffolds was assessed before and after HA coating and uniform distribution of HA coating on the surface was observed by scanning electron microscopy. The scaffolds were implanted into rabbit femoral unicortical bone defects according to four treatment groups based on pore structure and HA coating. After 6 and 12 weeks, scaffolds and host bone were recovered and processed for histology. Data suggest that all configurations of the scaffolds integrated with the host bone and were biocompatible and thus may offer an exciting new scaffold platform for delivery of biologicals for bone regeneration.

Fiber-reinforced calcium phosphate cement formulations for cranioplasty applications: a 52-week duration preclinical rabbit calvaria study.

The in vivo tissue response to a newly developed fiber-reinforced calcium phosphate cement (CPC) formulation was assessed using a well-established rabbit calvarial defect model. Bilateral subcritical sized (8-mm diameter) defects were surgically created in the parietal bones of each rabbit (a total of 48 rabbits), and randomized to be filled with either the new fiber-reinforced formulation, a conventional CPC (positive control), or left unfilled (negative control). The implant sites were subsequently retrieved after 12, 24, and 52 weeks postsurgery. Each specimen, including the parietal bone craniotomy and underlying brain, were recovered at necropsy and the tissue responses were assessed by histology. The resulting histological slides indicated that there was no evidence of severe inflammatory responses or osteolysis. The data showed new dural and pericranial bone formation along the implants, as well as excellent bone-to-implant interfaces in all of the CPC-filled defects. These results suggest that the biologic response to the new fiber-reinforced CPC formulations and conventional nonreinforced CPC are very similar, and both demonstrate excellent biocompatibility as well as an overall osteophylic response.