Recent progress in regenerative therapy for damaged salivary glands: From bench to bedside.

OBJECTIVE: For functional restoration of salivary glands (SGs) injured by radiation therapy or Sjögren's syndrome (SS), various experimental approaches, such as gene therapy, tissue engineering, and cell-based therapy, have been proposed. This narrative review summarized recent progresses in research using cell-based therapies, including promising trials that could lead to bench-to-clinic applications. METHODS: A literature review based on PubMed publications in the last two decades was performed to summarize progresses in cell-based therapies for SG dysfunction. RESULTS: Over 100 experimental studies have shown the therapeutic potential of several types of cells, such as SG stem cells and mesenchymal stem cells, as well as effectively conditioned mononuclear cells, in both radiation injury and SS animal models. These therapies affect to slow fibrosis progression and stimulate tissue regeneration in atrophic glands. However, to date, only a total of seven studies have been developed to the stage of clinical study, showing the safety and preliminary efficacy. CONCLUSION: To lead the radical effectiveness expected in cell-based therapy, advances in reverse translational research and in innovative experimental research, based on the findings of recent clinical studies, will be critical in the next decade.

A Prospective Study of the Assessment of the Efficacy of a Biodegradable Poly(l-lactic acid/ε-caprolactone) Membrane for Guided Bone Regeneration.

Biodegradable guided bone regeneration (GBR) membranes consist primarily of collagen and aliphatic polyesters. This study assessed the comparative efficacy of a poly(l-lactic-caprolactone) [P(LA/CL)] membrane versus that of a collagen membrane in GBR. Patients requiring GBR simultaneously or before dental implant placement in edentulous regions were randomly assigned to one of two membranes. Within each membrane, they were subdivided into 3 groups: dental implants were placed simultaneously with GBR in groups A and B, and 180 days post-GBR in group C. The augmented bone width was measured at 1, 3, and 6 mm from the implant's neck (groups A and B) or the reference line (group C), utilizing cone-beam computed tomography images, immediately and 150 days post-surgery. A histological study was performed to evaluate bone formation in group C. No adverse events were observed. In the collagen group, the absorbed ratios of the augmented bone were 40.9 ± 36.7%, 29.4 ± 30.1%, and 11.1 ± 22.0% at 1, 3, and 6 mm, respectively; the ratio at 6 mm was significantly lower than that at 1 mm (p = 0.0442). In the P(LA/CL) group, those were 26.2 ± 27.3%, 17.1 ± 19.7%, and 13.3 ± 16.4% at 1, 3, and 6 mm, respectively, with no significant difference at each point. No significant inter-membrane differences were observed. The bone augmentation potential of the P(LA/CL) membrane matched that of the collagen membrane. P(LA/CL) could be used as a safe and effective membrane in GBR.

Immunomodulatory Macrophages Enable E-MNC Therapy for Radiation-Induced Salivary Gland Hypofunction.

A newly developed therapy using effective-mononuclear cells (E-MNCs) is reportedly effective against radiation-damaged salivary glands (SGs) due to anti-inflammatory and revascularization effects. However, the cellular working mechanism of E-MNC therapy in SGs remains to be elucidated. In this study, E-MNCs were induced from peripheral blood mononuclear cells (PBMNCs) by culture for 5-7 days in medium supplemented with five specific recombinant proteins (5G-culture). We analyzed the anti-inflammatory characteristics of macrophage fraction of E-MNCs using a co-culture model with CD3/CD28-stimulated PBMNCs. To test therapeutic efficacy in vivo, either E-MNCs or E-MNCs depleted of CD11b-positive cells were transplanted intraglandularly into mice with radiation-damaged SGs. Following transplantation, SG function recovery and immunohistochemical analyses of harvested SGs were assessed to determine if CD11b-positive macrophages contributed to tissue regeneration. The results indicated that CD11b/CD206-positive (M2-like) macrophages were specifically induced in E-MNCs during 5G-culture, and Msr1- and galectin3-positive cells (immunomodulatory macrophages) were predominant. CD11b-positive fraction of E-MNCs significantly inhibited the expression of inflammation-related genes in CD3/CD28-stimulated PBMNCs. Transplanted E-MNCs exhibited a therapeutic effect on saliva secretion and reduced tissue fibrosis in radiation-damaged SGs, whereas E-MNCs depleted of CD11b-positive cells and radiated controls did not. Immunohistochemical analyses revealed HMGB1 phagocytosis and IGF1 secretion by CD11b/Msr1-positive macrophages from both transplanted E-MNCs and host M2-macrophages. Thus, the anti-inflammatory and tissue-regenerative effects observed in E-MNC therapy against radiation-damaged SGs can be partly explained by the immunomodulatory effect of M2-dominant macrophage fraction.

Effective-mononuclear cell (E-MNC) therapy alleviates salivary gland damage by suppressing lymphocyte infiltration in Sjögren-like disease.

Introduction: Sjögren syndrome (SS) is an autoimmune disease characterized by salivary gland (SG) destruction leading to loss of secretory function. A hallmark of the disease is the presence of focal lymphocyte infiltration in SGs, which is predominantly composed of T cells. Currently, there are no effective therapies for SS. Recently, we demonstrated that a newly developed therapy using effective-mononuclear cells (E-MNCs) improved the function of radiation-injured SGs due to anti-inflammatory and regenerative effects. In this study, we investigated whether E-MNCs could ameliorate disease development in non-obese diabetic (NOD) mice as a model for primary SS. Methods: E-MNCs were obtained from peripheral blood mononuclear cells (PBMNCs) cultured for 7 days in serum-free medium supplemented with five specific recombinant proteins (5G culture). The anti-inflammatory characteristics of E-MNCs were then analyzed using a co-culture system with CD3/CD28-stimulated PBMNCs. To evaluate the therapeutic efficacy of E-MNCs against SS onset, E-MNCs were transplanted into SGs of NOD mice. Subsequently, saliva secretion, histological, and gene expression analyses of harvested SG were performed to investigate if E-MNCs therapy delays disease development. Results: First, we characterized that both human and mouse E-MNCs exhibited induction of CD11b/CD206-positive cells (M2 macrophages) and that human E-MNCs could inhibit inflammatory gene expressions in CD3/CD28- stimulated PBMNCs. Further analyses revealed that Msr1-and galectin3-positive macrophages (immunomodulatory M2c phenotype) were specifically induced in E-MNCs of both NOD and MHC class I-matched mice. Transplanted E-MNCs induced M2 macrophages and reduced the expression of T cell-derived chemokine-related and inflammatory genes in SG tissue of NOD mice at SS-onset. Then, E-MNCs suppressed the infiltration of CD4-positive T cells and facilitated the maintenance of saliva secretion for up to 12 weeks after E-MNC administration. Discussion: Thus, the immunomodulatory actions of E-MNCs could be part of a therapeutic strategy targeting the early stage of primary SS.

First-in-Human Study to Investigate the Safety Assessment of Peri-Implant Soft Tissue Regeneration with Micronized-Gingival Connective Tissue: A Pilot Case Series Study.

Background: We have recently proposed an alternative strategy of free gingival graft (FGG) and connective tissue graft (CTG) using micronized-gingival connective tissues (MGCTs). The advantage of this strategy is that MGCTs from a small piece of maxillary tuberosity can regenerate the keratinized tissue band. However, safety and efficacy have not yet been established in patients. This clinical study was a pilot case series, and the objective was to assess the safety and the preliminary efficacy of MGCTs on peri-implant mucosa regeneration. Methods: This was a pilot interventional, single-center, first-in-human (FIH), open (no masking), uncontrolled, and single-assignment study. A total of 4 patients who needed peri-implant soft tissues reconstruction around dental implants received transplantation of atelocollagen-matrix with MGCTs micronized by the tissue disruptor technique. The duration of intervention was 4 weeks after surgery. Results: This first clinical study demonstrated that using MGCTs did not cause any irreversible adverse events, and it showed the preliminary efficacy for peri-implant soft tissues reconstruction in dental implant therapy. Conclusions: Though further studies are needed on an appropriate scale, as an alternative strategy of FGG or CTG, MGCTs might be promising for peri-implant mucosa reconstruction without requiring a high level of skills and morbidity to harvest graft tissues.

Clinical Outcome and 8-Year Follow-Up of Alveolar Bone Tissue Engineering for Severely Atrophic Alveolar Bone Using Autologous Bone Marrow Stromal Cells with Platelet-Rich Plasma and ß-Tricalcium Phosphate Granules.

BACKGROUND: Although bone tissue engineering for dentistry has been studied for many years, the clinical outcome for severe cases has not been established. Furthermore, there are limited numbers of studies that include long-term follow-up. In this study, the safety and efficacy of bone tissue engineering for patients with a severely atrophic alveolar bone were examined using autogenous bone marrow stromal cells (BMSCs), and the long-term stability was also evaluated. METHODS: BMSCs from iliac bone marrow aspirate were cultured and expanded. Then, induced osteogenic cells were transplanted with autogenous platelet-rich plasma (PRP) and ß-tricalcium phosphate granules (ß-TCP) for maxillary sinus floor and alveolar ridge augmentation. Eight patients (two males and six females) with an average age of 54.2 years underwent cell transplantation. Safety was assessed by monitoring adverse events. Radiographic evaluation and bone biopsies were performed to evaluate the regenerated bone. RESULTS: The major population of transplanted BMSCs belonged to the fraction of CD34-, CD45dim, and CD73+ cells, which was only 0.065% of the total bone marrow cells. Significant deviations were observed in cell growth and alkaline phosphatase activities among individuals. However, bone regeneration was observed in all patients and the average bone area in the biopsy samples was 41.9% 6 months following transplantation, although there were also significant deviations among each case. No adverse events related to the transplants were observed. In the regenerated bone, 27 out of 29 dental implants were integrated. Dental implants and regenerated bone were stable for an average follow-up period of 7 years and 10 months. CONCLUSIONS: Although individual variations were observed, the results showed that bone tissue engineering using BMSCs with PRP and ß-TCP was feasible for patients with severe atrophic maxilla throughout a long-term follow-up period and was considered safe. However, further studies with a larger number of cases and controls to confirm the efficacy of BMSCs and the development of a protocol to establish a reproducible quality of stem cell-based graft material will be required.

Clinical Safety Assessment of Autologous Freeze-Drying Platelet-Rich Plasma for Bone Regeneration in Maxillary Sinus Floor Augmentation: A Pilot Study.

The purpose of this clinical study is to evaluate the safety and preliminary efficacy of autologous freeze-drying platelet-rich plasma (FD-PRP) on bone regeneration in maxillary sinus floor augmentation as a preliminary pilot study. Five patients that required sinus floor augmentation to facilitate the placement of dental implants participated in this clinical study. The PRP was prepared from the autologous peripheral blood and was lyophilized and stored at -20 °C for 4 weeks before surgery. At surgery, triple-concentrated FD-PRP (x3FD-PRP) mixed with synthetic bone grafting materials was rehydrated following the transplantation into the sinus floor. The primary outcome was a safety verification of x3FD-PRP, evaluated in terms of the clinical course and consecutive blood tests. The secondary outcome was clinical efficacy focused on bone regeneration in sinus floor augmentation evaluated by radiographic examination and implant stability. There were no adverse events, such as systemic complications, excessive inflammatory reactions, severe infection, or local site healing complications, besides those on the usual course associated with surgery. Vertical augmented height was maintained, and the initial stability of implants was achieved post-operatively in 6 months. The results obtained in this study suggest that x3FD-PRP can be used safely for bone engineering in clinical practice. Further studies are required to draw a conclusion concerning the efficacy of x3FD-PRP since this was a pilot study with a single arm and a small sample size.

Resident CD34-positive cells contribute to peri-endothelial cells and vascular morphogenesis in salivary gland after irradiation.

Salivary gland (SG) hypofunction is a common post-radiotherapy complication. Besides the parenchymal damage after irradiation (IR), there are also effects on mesenchymal stem cells (MSCs) which were shown to contribute to regeneration and repair of damaged tissues by differentiating into stromal cell types or releasing vesicles and soluble factors supporting the healing processes. However, there are no adequate reports about their roles during SG damage and regeneration so far. Using an irradiated SG mouse model, we performed certain immunostainings on tissue sections of submandibular glands at different time points after IR. Immunostaining for CD31 revealed that already one day after IR, vascular impairment was induced at the level of capillaries. In addition, the expression of CD44-a marker of acinar cells-diminished gradually after IR and, by 20 weeks, almost disappeared. In contrast, the number of CD34-positive cells significantly increased 4 weeks after IR and some of the CD34-positive cells were found to reside within the adventitia of arteries and veins. Laser confocal microscopic analyses revealed an accumulation of CD34-positive cells within the area of damaged capillaries where they were in close contact to the CD31-positive endothelial cells. At 4 weeks after IR, a fraction of the CD34-positive cells underwent differentiation into α-SMA-positive cells, which suggests that they may contribute to regeneration of smooth muscle cells and/or pericytes covering the small vessels from the outside. In conclusion, SG-resident CD34-positive cells represent a population of progenitors that could contribute to new vessel formation and/or remodeling of the pre-existing vessels after IR and thus, might be an important player during SG tissue healing.

Phase 1 clinical study of cell therapy with effective-mononuclear cells (E-MNC) for radiogenic xerostomia (first-in-human study) (FIH study on E-MNC therapy for radiogenic xerostomia).

BACKGROUND: Treatment for most patients with head and neck cancers includes ionizing radiation with or without chemotherapy. This treatment causes irreversible damage to salivary glands in the irradiation field accompanied by a loss of fluid-secreting acinar cells and a considerable decrease of saliva secretion. There is currently no adequate conventional treatment for this condition. In recent years, we developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs), and such effectively conditioned PBMNC (E-MNC) therapy has shown promising improvements to the function of radiation-injured salivary glands in preclinical studies. However, the safety and effect of E-NMC therapy have yet assessed in human. The objective of this ongoing first-in-man study is to assess the safety, tolerability, and in part the efficacy of E-MNC therapy for treating radiation-induced xerostomia. METHODS/DESIGN: This phase 1 first-in-man study is an open-label, single-center, two-step dose escalation study. A total of 6 patients, who had no recurrence of head and neck cancer over 5 years following radiation therapy and suffered from radiation-induced xerostomia, will receive a transplantation of E-NMCs derived from autologous PBMNCs to a submandibular gland. The duration of the intervention will be 1 year. To analyze the recovery of salivary secretion, a gum test will be performed. To analyze the recovery of atrophic salivary glands, computed tomography (CT), and magnetic resonance imaging (MRI) of salivary glands will be conducted. The primary endpoint is the safety of the protocol. The secondary endpoints are the changes from baseline in whole saliva secretion and salivary gland atrophy. DISCUSSION: This will be the first clinical study of regenerative therapy using E-MNCs for patients with severe radiation-induced xerostomia. The results of this study are expected to contribute to developing the low-invasive cell-based therapy for radiation-induced xerostomia. TRIAL REGISTRATION: This study was registered with the Japan Registry of Clinical Trials (http://jrct.niph.go.jp) as jRCTb070190057.

Do not keep it simple: recent advances in the generation of complex organoids.

3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models.

First clinical application of octacalcium phosphate collagen composite on bone regeneration in maxillary sinus floor augmentation: A prospective, single-arm, open-label clinical trial.

The overall objective of this study was to assess the safety and efficacy of OCP/Col as a bone substitute material for bone regeneration during sinus floor augmentation. Maxillary sinus floor augmentation was performed thorough lateral window approach. According to the height of host bone, simultaneous approach (≥5 mm) or staged approach (less than 5 mm) was applied. In this research, clinical findings of dental implant treatment after setting the restorations were set as a primary endpoint in both approaches (infection, inflammation around the implant, movement of the implant, pain, sensory disorder, and bone resorption around the implant body on radiological evaluation.). In staged approach, histological evaluation of bone biopsy specimen was also conducted. As secondary endpoints, hounsfield unit (HU) value, vertical bone height, implant stability quotient (ISQ), and adverse events during the research were evaluated. In all cases, as a primary endpoint, clinical findings after setting the restorations were uneventful with no adverse events. Histological structure demonstrated mature bone derived from OCP/Col. In the ossified area, osteogenesis was observed around OCP granules, and osteoblast-like cells were arrayed around OCP granules. Osteocyte encapsulation was recognized in the new bone. HU increased over time with both approaches. Vertical bone height significantly increased at 3 months postoperatively, and maintained during follow-up. ISQ increased with both approaches. In particular, ISQ was significantly increased with the staged approach. This clinical trial demonstrated the safety and efficacy of OCP/Col for bone regeneration in maxillary sinus floor augmentation. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 108B:243-252, 2020.

Intra-Bone Marrow Administration of Mesenchymal Stem/Stromal Cells Is a Promising Approach for Treating Osteoporosis.

Mesenchymal stem/stromal cells (MSCs) are known to be useful for treating local bone diseases. However, it is not known if MSCs are effective for treating systemic bone diseases, as the risk for mortality following intravenous MSC administration has hindered research progress. In this study, we compared the safety and efficacy of intra-bone marrow and intravenous administration of MSCs for the treatment of ovariectomy- (OVX-) induced osteoporosis. Cells capable of forming bone were isolated from the murine compact bones and expanded in culture. Relatively pure MSCs possessing increased potential for cell proliferation, osteogenic differentiation, and inhibition of osteoclastogenesis were obtained by magnetic-activated cell sorting with the anti-Sca-1 antibody. Sca-1-sorted MSCs were administered to OVX mice, which were sacrificed 1 month later. We observed that 22% of the mice died after intravenous administration, whereas none of the mice died after intra-bone marrow administration. With respect to efficacy, intravenous administration improved bone mineral density (BMD) by increasing bone mineral content without affecting bone thickness, whereas intra-bone marrow administration improved BMD by increasing both bone mineral content and bone thickness. These results indicate that intra-bone marrow administration of pure MSCs is a safer and more effective approach for treating osteoporosis.

Anti-inflammatory and vasculogenic conditioning of peripheral blood mononuclear cells reinforces their therapeutic potential for radiation-injured salivary glands.

BACKGROUND: There are currently no effective treatments available for patients with irreversible loss of salivary gland (SG) function caused by radiation therapy for head and neck cancer. In this study, we have developed an effective culture method to enhance the anti-inflammatory and vasculogenic phenotypes of peripheral blood mononuclear cells (PBMNCs) and investigated whether such effectively conditioned PBMNCs (E-MNCs) could regenerate radiation-injured SGs and ameliorate salivary secretory function in mice. METHODS: Mouse PBMNCs were expanded in primary serum-free culture with five vasculogenic proteins for 5 days, and then the resulting cells (E-MNCs) were analyzed for their characteristics. Subsequently, 5 × 104 E-MNCs (labeled with EGFP in some experiments) were injected intra-glandularly into a mouse model of radiation-injured atrophic submandibular glands. After 2-3 weeks, the submandibular glands were harvested, and then the injected E-MNCs were tracked. Four, 8, and 12 weeks after irradiation (IR), salivary outputs were measured to evaluate the recovery of secretory function, and the gland tissues were harvested for histological and gene expression analyses to clarify the effects of cell transplantation. RESULTS: The resulting E-MNCs contained an enriched population of definitive CD11b/CD206-positive (M2 macrophage-like) cells and showed anti-inflammatory and vasculogenic characteristics. Salivary secretory function in E-MNC-transplanted mice gradually recovered after 4 weeks post-irradiation (post-IR) and reached 3.8-fold higher than that of non-transplanted mice at 12 weeks. EGFP-expressing E-MNCs were detected in a portion of the vascular endothelium and perivascular gland tissues at 2 weeks post-IR, but mainly in some microvessels at 3 weeks. Between 4 and 12 weeks post-IR, mRNA expression and histological analyses revealed that E-MNC transplantation reduced the expression of inflammatory genes and increased the level of tissue-regenerative activities such as stem cell markers, cell proliferation, and blood vessel formation. At 12 weeks post-IR, the areas of acinar and ductal cells regenerated, and the glands had less fibrosis. CONCLUSIONS: This effective conditioning of PBMNCs is a simple, rapid, and efficient method that provides a non-invasive source of therapeutic cells for regenerating radiation-injured atrophic SGs.

Systemic administration of quality- and quantity-controlled PBMNCs reduces bisphosphonate-related osteonecrosis of jaw-like lesions in mice.

BACKGROUND: Definitive treatment strategies for bisphosphonate-related osteonecrosis of the jaw (BRONJ) have not been developed. Cell-based therapy is an attractive treatment method for intractable diseases in the medical and dental fields; however, approval has been challenging in dentistry. Recently, we developed quality- and quantity (QQ)-controlled peripheral blood mononuclear cells (PBMNCs) that have anti-inflammatory and pro-angiogenesis effects. The aim of this study was to investigate the effects of QQ-controlled PBMNC transplantation on BRONJ-like lesions in mice. METHODS: To create high-prevalence BRONJ-like lesions, cyclophosphamide (CY) and zoledronate (ZA) were used with tooth extraction. Drug treatment was performed for 5 weeks. QQ-controlled PBMNC transplantation was performed immediately following tooth extraction of both maxillary first molars at 3 weeks after drug administration. Mice were euthanized at 2 weeks post-extraction. Histomorphometric and immunohistochemical analyses, microcomputed tomography assessment, and quantitative polymerase chain reaction evaluation were conducted using maxillae and long bones. RESULTS: ZA effects on long bones were noted, regardless of CY. Severely inhibited osseous and soft tissue wound healing of tooth extraction sockets was induced by CY/ZA combination therapy, which was diagnosed as BRONJ-like lesions. QQ-controlled PBMNC transplantation reduced BRONJ-like lesions by improving soft tissue healing with increased M1 and M2 macrophages and enhanced neovascularization in the connective tissue of tooth extraction sockets. QQ-controlled PBMNC transplantation also reduced inflammation by decreasing polymorphonuclear cells and TNF-α expression in the tooth extraction sockets. Additionally, QQ-controlled PBMNC transplantation partially improved osseous healing of tooth extraction sockets. Interestingly, only 20,000 QQ-controlled PBMNCs per mouse induced these transplantation effects. QQ-controlled PBMNC transplantation did not affect the systemic microenvironment. CONCLUSIONS: Our findings suggest that transplantation of a small amount of QQ-controlled PBMNCs may become novel therapeutic or prevention strategies for BRONJ without any adverse side effects.

Alveolar bone preservation by a hydroxyapatite/collagen composite material after tooth extraction.

OBJECTIVE: The aim of this study was to assess the effectiveness of a hydroxyapatite/collagen composite material (HAp/Col) for preservation of alveolar bone after tooth extraction. MATERIALS AND METHODS: HAp/Col was applied to the alveolus bone ridge preservation after tooth extraction, because of subsequent dental implant placement in 35 regions of 24 patients (mean age, 59.3 years; range, 25-81 years). Cone beam computed tomography was used to assess changes in alveolar bone at the extraction site before and at 3 months (mean, 13.7 weeks; range, 10-17 weeks) after tooth extraction. Changes in height and width of the alveolar bone were measured to evaluate bone reduction after surgery. Bone biopsy was performed at 11 regions of dental implant placement to observe bone regeneration and remaining material in the extraction socket. RESULTS: The alveolar bone height was decreased by 0.00 ± 2.44 mm at the buccal side and 0.35 ± 1.73 mm at the lingual side, while the width was decreased by 1.02 ± 1.64 mm at 3 months after surgery. The middle of the socket floor was elevated by 5.71 ± 3.45 mm at 3 months after surgery. Bone biopsy specimens revealed no remaining implanted material, and approximately 49.79 ± 14.41% of the specimens were occupied by bone tissue. CONCLUSIONS: According to the result of this study, HAp/Col is a reliable material to presearve alveolar bone after tooth extraction. CLINICAL RELEVANCE: HAp/Col contributes dental implant treatment due to maintain the alveolar bone after tooth extraction.

Bone marrow concentrate promotes bone regeneration with a suboptimal-dose of rhBMP-2.

Bone marrow concentrate (BMC), which is enriched in mononuclear cells (MNCs) and platelets, has recently attracted the attention of clinicians as a new optional means for bone engineering. We previously reported that the osteoinductive effect of bone morphogenetic protein-2 (BMP-2) could be enhanced synergistically by co-transplantation of peripheral blood (PB)-derived platelet-rich plasma (PRP). This study aims to investigate whether BMC can effectively promote bone formation induced by low-dose BMP-2, thereby reducing the undesirable side-effects of BMP-2, compared to PRP. Human BMC was obtained from bone marrow aspirates using an automated blood separator. The BMC was then seeded onto ß-TCP granules pre-adsorbed with a suboptimal-dose (minimum concentration to induce bone formation at 2 weeks in mice) of recombinant human (rh) BMP-2. These specimens were transplanted subcutaneously to the dorsal skin of immunodeficient-mice and the induction of ectopic bone formation was assessed 2 and 4 weeks post-transplantation. Transplantations of five other groups [PB, PRP, platelet-poor plasma (PPP), bone marrow aspirate (BM), and BM-PPP] were employed as experimental controls. Then, to clarify the effects on vertical bone augmentation, specimens from the six groups were transplanted for on-lay placement on the craniums of mice. The results indicated that BMC, which contained an approximately 2.5-fold increase in the number of MNCs compared to PRP, could accelerate ectopic bone formation until 2 weeks post-transplantation. On the cranium, the BMC group promoted bone augmentation with a suboptimal-dose of rhBMP-2 compared to other groups. Particularly in the BMC specimens harvested at 4 weeks, we observed newly formed bone surrounding the TCP granules at sites far from the calvarial bone. In conclusion, the addition of BMC could reduce the amount of rhBMP-2 by one-half via its synergistic effect on early-phase osteoinduction. We propose here that BMC transplantation facilitates the clinical use of rhBMP-2 as an alternative strategy for bone engineering.

Optimal timing and frequency of bone marrow soup therapy for functional restoration of salivary glands injured by single-dose or fractionated irradiation.

Injections of bone marrow (BM) cell extract, known as 'BM soup', were previously reported to mitigate ionizing radiation (IR) injury to salivary glands (SGs). However, the optimal starting time and frequency to maintain BM soup therapeutic efficacy remains unknown. This study tested the optimal starting time and frequency of BM soup injections in mice radiated with either a single dose or a fractionated dose. First, BM soup treatment was started at 1, 3 or 7 weeks post-IR; positive (non-IR) and negative (IR) control mice received injections of saline (vehicle control). Second, BM soup-treated mice received injections at different frequencies (1, 2, 3 and 5 weekly injections). Third, a 'fractionated-dose radiation' model to injure mouse SGs was developed (5 Gy × 5 days) and compared with the single high dose radiation model. All mice (n = 65) were followed for 16 weeks post-IR. The results showed that starting injections of BM soup between 1 and 3 weeks mitigated the effect of IR-induced injury to SGs and improved the restoration of salivary function. Although the therapeutic effect of BM soup lessens after 8 weeks, it can be sustained by increasing the frequency of weekly injections. Moreover, both single-dose and fractionated-dose radiation models are efficient and comparable in inducing SG injury and BM soup treatments are effective in restoring salivary function in both radiation models. In conclusion, starting injections of BM soup within 3 weeks post-radiation, with 5 weekly injections, maintains 90-100% of saliva flow in radiated mice.

A Simplified and Systematic Method to Isolate, Culture, and Characterize Multiple Types of Human Dental Stem Cells from a Single Tooth.

This chapter describes a simplified method that allows the systematic isolation of multiple types of dental stem cells such as dental pulp stem cells (DPSC), periodontal ligament stem cells (PDLSC), and stem cells of the apical papilla (SCAP) from a single tooth. Of specific interest is the modified laboratory approach to harvest/retrieve the dental pulp tissue by minimizing trauma to DPSC by continuous irrigation, reduction of frictional heat from the bur rotation, and reduction of the bur contact time with the dentin. Also, the use of a chisel and a mallet will maximize the number of live DPSC for culture. Steps demonstrating the potential for multiple cell differentiation lineages of each type of dental stem cell into either osteocytes, adipocytes, or chondrocytes are described. Flow cytometry, with a detailed strategy for cell gating and analysis, is described to verify characteristic markers of human mesenchymal multipotent stromal cells (MSC) from DPSC, PDLSC, or SCAP for subsequent experiments in cell therapy and in tissue engineering. Overall, this method can be adapted to any laboratory with a general setup for cell culture experiments.

Efficacy of freeze-dried platelet-rich plasma in bone engineering.

OBJECTIVE: Platelet-rich plasma (PRP) is typically isolated and applied immediately after preparation, making it both a time- and labor-intensive addition to the operative procedure. Thus, it would be convenient if PRP could be preserved. We evaluated the efficacy of freeze-dried PRP (FD-PRP), as compared with freshly isolated PRP (f-PRP) for bone engineering. DESIGN: FD-PRP was prepared by lyophilization of f-PRP and was subsequently preserved at -20°C for one month. It was then rehydrated with an equal or 1/3 amount of distilled water (×1FD-PRP, ×3FD-PRP, respectively), and we assessed its gelation properties and the release of growth factors (PDGF-BB, TGF-ß1, and VEGF). We also examined the bone forming ability with onlay-grafting on mice calvaria using ß-TCP granules as a scaffold. RESULTS: FD-PRP showed comparable gelation as f-PRP. In terms of growth factor release,×1FD-PRP released identical concentrations of PDGF-BB and TGF-ß1 to f-PRP, while ×3FD-PRP released approximately 3-fold concentrations when compared with f-PRP. In vivo, ×1FD-PRP promoted identical levels of the bone formation as f-PRP, and ×3FD-PRP induced more abundant bone formation. CONCLUSIONS: These results suggest that f-PRP can be stored without functional loss by freeze-drying and the concentration of PRP may improve its efficacy in bone engineering.

Onlay bone augmentation on mouse calvarial bone using a hydroxyapatite/collagen composite material with total blood or platelet-rich plasma.

OBJECTIVE: The aim of this study was to assess newly formed onlay bone on mouse calvarial bone using a new artificial bone material, a hydroxyapatite/collagen composite, with total blood or platelet-rich plasma. DESIGN: The hydroxyapatite/collagen composite material with normal saline, total blood or platelet-rich plasma was transplanted on mouse calvarial bone. The mice were sacrificed and the specimens were harvested four weeks after surgery. The newly formed bone area was measured on hematoxylin and eosin stained specimens using Image J software. RESULTS: The hydroxyapatite/collagen composite materials with total blood or platelet-rich plasma induced a significantly greater amount of newly formed bone than that with normal saline. Moreover, bone marrow was observed four weeks after surgery in the transplanted materials with total blood or platelet-rich plasma but not with normal saline. However, there were no significant differences in the amount of newly formed bone between materials used with total blood versus platelet-rich plasma. CONCLUSIONS: The hydroxyapatite/collagen composite material was valid for onlay bone augmentation and this material should be soaked in total blood or platelet-rich plasma prior to transplantation.