Dongdong Fang

Identification of the active components in Bone Marrow Soup: a mitigator against irradiation-injury to salivary glands

In separate studies, an extract of soluble intracellular contents from whole bone marrow cells, named “Bone Marrow (BM) Soup”, was reported to either improve cardiac or salivary functions post-myocardial infarction or irradiation (IR), respectively. However, the active components in BM Soup are unknown. To demonstrate that proteins were the active ingredients, we devised a method using proteinase K followed by heating to deactivate proteins and for safe injections into mice. BM Soup and “deactivated BM Soup” were injected into mice that had their salivary glands injured with 15Gy IR. Control mice received either injections of saline or were not IR. Results at week 8 post-IR showed the ‘deactivated BM Soup’ was no better than injections of saline, while injections of native BM Soup restored saliva flow, protected salivary cells and blood vessels from IR-damage. Protein arrays detected several angiogenesis-related factors (CD26, FGF, HGF, MMP-8, MMP-9, OPN, PF4, SDF-1) and cytokines (IL-1ra, IL-16) in BM Soup. In conclusion, the native proteins (but not the nucleic acids, lipids or carbohydrates) were the therapeutic ingredients in BM Soup for functional salivary restoration following IR. This molecular therapy approach has clinical potential because it is theoretically less tumorigenic and immunogenic than cell therapies.

Three-Dimensional Printed Scaffolds with Multipotent Mesenchymal Stromal Cells for Rabbit Mandibular Reconstruction and Engineering

Multipotent mesenchymal stromal cells (MSC) derived from both the bone marrow and adipose tissue possess the ability to differentiate into multiple cell lineages, regulate the immune function by secreting numerous bioactive paracrine factors, and hold great potential in cell therapy and tissue engineering. When combined with three-dimensional (3D) scaffolds, MSC can be used for bone defect reconstruction and engineering. This protocol describes the isolation of bone marrow mesenchymal stromal cells (BMMSC) and adipose-tissue derived stem cells (ADSC) from rabbits for subsequent seeding on tissue-engineered 3D-printed scaffolds and transplantation into a rabbit-model with the goal of repairing large osseous mandibular defects (one quarter of the lower jaw is removed surgically). Steps to demonstrate the three cell differentiation lineage potentials of BMMSC and ADSC into osteocytes, adipocytes, and chondrocytes are described. A modified cell seeding method using syringes on scaffold is detailed. Creating a large mandibular bone defect, the rapid prototyping method to print a customized 3D-scaffold, the scaffold implantation procedure in rabbits, and microcomputed tomography (micro-CT) analysis are also described.

Scaffolds for epithelial tissue engineering customized in elastomeric molds

Restoration of soft tissue defects remains a challenge for surgical reconstruction. In this study, we introduce a new approach to fabricate poly(d,l-lactic acid) (PDLLA) scaffolds with anatomical shapes customized to regenerate three-dimensional soft tissue defects. Highly concentrated polymer/salt mixtures were molded in flexible polyether molds. Microcomputed tomography showed that with this approach it was possible to produce scaffolds with clinically acceptable volume ratio maintenance (>90%). Moreover, this technique allowed us to customize the average pore size and pore interconnectivity of the scaffolds by using variations of salt particle size. In addition, this study demonstrated that with the increasing porosity and/or the decreasing of the average pore size of the PDLLA scaffolds, their mechanical properties decrease and they degrade more slowly. Cell culture results showed that PDLLA scaffolds with an average pore size of 100 µm enhance the viability and proliferation rates of human gingival epithelial cells up to 21 days. The simple method proposed in this article can be extended to fabricate porous scaffolds with customizable anatomical shapes and optimal pore structure for epithelial tissue engineering.

Compact Bone-Derived Multipotent Mesenchymal Stromal Cells (MSCs) for the Treatment of Sjogren’s-like Disease in NOD Mice

Compact bone (cortical or dense bone) is among the organs that contain multipotent mesenchymal stromal cells (MSCs). Unlike bone marrow plugs where MSCs were initially isolated, compact bone has minimal (amount of) hematopoietic cells and thus facilitates the MSCs isolation process. In vitro, MSCs from compact bone show multipotency and differentiation into mesenchymal tissues such as bone, adipose, and cartilage, under certain conditions. MSCs therapy has been promising in preclinical and clinical studies against autoimmune diseases. Not only can MSCs replace the lost tissue through their regenerative properties, but they can also control the autoimmune attacks by immunoregulatory cytokines. This protocol describes the use of compact bone-derived MSCs to preserve salivary function (saliva flow/output) in the NOD (nonobese diabetic) mouse model affected with Sjogrens-like disease.

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.

Three-dimensionally printed polyetherketoneketone scaffolds with mesenchymal stem cells for the reconstruction of critical-sized mandibular defects

Additive manufacturing offers a tailored approach to tissue engineering by providing anatomically precise scaffolds onto which stem cells and growth factors can be supplied. Polyetherketoneketone (PEKK), an ideal candidate biomaterial, is limited by a poor implant–bone interface but can be functionalized with adipose‐derived stem cells (ADSC) to promote integration. This in vivo study examined the interaction of a three‐dimensional printed PEKK/ADSC implant within the critical‐sized mandibular defect in a rabbit model.

Lyophilized bone marrow cell extract functionally restores irradiation-injured salivary glands

OBJECTIVE Bone marrow cell extract (BMCE) was previously reported to restore salivary gland hypofunction caused by irradiation injury. Proteins were shown to be the main active factors in BMCE. However, BMCE therapy requires multiple injections and protein denaturation is a concern during BMCE storage. This study aimed to preserve, by lyophilization (freeze-drying), the bioactive factors in BMCE. METHODS We developed a method to freeze-dry BMCE and then to analyze its ingredients and functions in vivo. Freeze-dried (FD) BMCE, freshly prepared BMCE (positive control), or saline (vehicle control) was injected into the tail vein of mice that had received irradiation to damage their salivary glands. RESULTS Results demonstrated that the presence of angiogenesis-related factors and cytokines in FD-BMCE remained comparable to those found in fresh BMCE. Both fresh and FD-BMCE restored comparably saliva secretion, increased cell proliferation, upregulated regenerative/repair genes, protected salivary acinar cells, parasympathetic nerves, and blood vessels from irradiation-damaged salivary glands. CONCLUSION Lyophilization of BMCE maintained its bioactivity and therapeutic effect on irradiation-injured salivary glands. The advantages of freeze-drying BMCE are its storage and transport at ambient temperature.

Cell extracts from spleen and adipose tissues restore function to irradiation-injured salivary glands.

A cell extract from whole bone marrow (BM), which we named “BM Soup,” has the property to restore saliva secretion to irradiation (IR)‐injured salivary glands (SGs). However, BM cell harvesting remains an invasive procedure for the donor. The main objective of this study was to test the therapeutic effect of “Cell Soups” obtained from alternate tissues, such as adipose‐derived stromal cells (ADSCs) and spleen cells to repair SGs. BM Soup, Spleen Soup, ADSC Soup, or saline (vehicle control) was injected intravenously into mice with IR‐injured SGs (13Gy). Results demonstrated that all three cell soups restored 65–70% of saliva secretion, protected acinar cells, blood vessels, and parasympathetic nerves, and increased cell proliferation. Although protein array assays identified more angiogenesis‐related growth factors in ADSC Soup, the length of its therapeutic efficiency on saliva flow was less than that of the BM Soup and Spleen Soup. Another objective of this study was to compare “Fresh” versus “Cryopreserved (−80 °C)” BM Soup. It was found that the therapeutic effect of 12‐month “Cryopreserved BM Soup” was comparable to that of “Fresh BM Soup” on the functional restoration of IR‐injured SGs. In conclusion, both Spleen Soup and ADSC Soup can be used to mitigate IR‐damaged SGs.

Adult Stem Cell Therapy for Salivary Glands, with a Special Emphasis on Mesenchymal Stem Cells

Mesenchymal stromal/stem cell (MSC) therapy with the goal of restoring salivary function following irradiation injury or in Sjogren’s syndrome (SS) has made significant advances within the past 5 years. The majority of studies used MSCs obtained from the bone marrow or adipose tissue, but MSCs isolated from the salivary gland, dental pulp, and umbilical cord also demonstrated a therapeutic efficacy in reestablishing salivary function. Based on the amount of stimulated saliva secretion as a functional quantitative measure, irradiated mice/rats that received MSC therapy restored their salivary flow rate (SFR) to 60–90 % of normal age-matched animals, while SFR of irradiated animals without treatment remained at 35–50 % of secretory function. Thus, there was 25–40 % therapeutic improvement in animals receiving MSC therapy versus those that did not. This would be clinically significant because patients with severe salivary hypofunction (dry mouth) due to head and neck irradiation have no improvement in SFR, if left untreated. In the SS-like disease mouse model, MSC therapy restored SFR 80–100 % when treatment was given at an initial phase of SS-like disease, while its effectiveness decreased to 50–60 % when given at an advanced stage of disease. In SS patients, MSC therapy improved SFR by 40–50 %. When tested in the rodent model, MSC therapy was successful in restoring/maintaining the gland normal weights and histology (acinar cells, blood vessels) and upregulated the expression of genes favorable for salivary gland development and regeneration while downregulating inflammation and cell apoptosis, promising positive effects of MSC therapy.