Oral plaque from Type 2 diabetic patients reduces the clonogenic capacity of dental pulp-derived mesenchymal stem cells

Type 2 diabetes (T2D) is a major metabolic disease and a key epigenetic risk factor for the development of additional clinical complications. Among them, periodontitis (PD), a severe inflammatory disease ascribable to a dysregulated physiology and composition of the oral microbiota, represents one of the most relevant complications. Periodontitis can impact the structure of the tooth and likely the stem and progenitor cell pool, which actively contributes to the periodontal microenvironment and homeostasis. Modifications of the oral plaque play a key role in the etiopathogenesis of PD caused by T2D. However, to what extent the biology of the progenitor pool is affected has still to be elucidated. In this short report, we aimed to explore the biological effects of oral plaque derived from T2D patients with PD in comparison to non-diabetic patients with PD. Oral plaque samples were isolated from T2D and non-diabetic subjects with PD. Dental pulp stem cells (DPSCs), derived from the premolar tooth, were conditioned for 21 days with oral plaque samples and tested for their clonogenic ability. Cultures were also induced to differentiate towards the osteogenic lineage, and ALP and osteocalcin gene expression levels were evaluated by real-time qPCR. Results have shown that the number of clones generated by DPSCs exposed to T2D oral plaque was significantly lower compared to controls (ctl). The multivariate analysis confirmed that the decreased clonogenesis was significantly correlated only with T2D diagnosis. Moreover, the effect of T2D oral plaque was specific to DPSCs. Indicators of osteogenic differentiation were not significantly affected. This study provides a new biological insight into the effects ascribable to T2D in PD

The Effects of Bone Morphogenic Protein 2 on the Viability and Proliferation of Dental Pulp Stem Cell Isolates

Growth factor is an umbrella term used to describe a signaling molecule present in organisms that serves the purpose of influencing a stimulatory or inhibitory response from the target cell it acts on. Growth factors are categorized and classified by the types of tissues they act on and the cellular responses they illicit. For example, there are growth factors that act primarily in connective tissue on fibroblasts and they are aptly classified as the fibroblast growth factor family. Similarly, there is another family of growth factors that act on bone forming cells, among others, and they are known as bone morphogenic proteins. One member from this group of growth factors, BMP-2, is the focus of the current research. Bone Morphogenic Proteins are part of the Transforming Growth Factor-beta superfamily of growth factors. Recently they have been implicated in a variety of pathological processes including colon cancer and Barrett’s esophagus. BMP-2 is known for its ability to stimulate bone formation and is the most successful bone modulator in the family. Its involvement in cellular differentiation into bone forming cells has been shown in many studies. BMP-2 acts on cell surface receptors which, through a series of reactions, enhances the likelihood of differentiating into a bone forming cell. This induction can be demonstrated in a variety of cell types. Those of particular interest are stem cells. There are three main types of stem cells including: embryonic stem cells, adult non- embryonic stem cells, induced pluripotent stem cells. Adult stem cells are the most abundant and are less controversial than embryonic stem cells. Currently adult stem cells are being isolated from a variety of tissues including those of mesenchymal tissue origin. The cell type in the current study are dental pulp stem cells (DPSC) which have been shown to display phenotypic changes in response to various growth factors. The use of multiple growth factors concomitantly on DPSC has not been studied in great depth, and thus it is not known if multiple stimulatory growth factors will act synergistically or antagonistically with one another. The data from the two following studies provide evidence that BMP-2 provides a stimulating influence on at least one dental pulp stem cell subset. When used concomitantly, BMP-2 and Vascular Endothelial Growth Factor (VEGF) enhances sDT DPSC phenotype (viability and growth) beyond that of either growth factor independently. Future studies may be needed to evaluate the potential for BMP-2 and other growth factors to induce DPSC differentiation and lineage-specific phenotypic changes for bioengineering applications or tissue regeneration

ZIF-8 Modified Polypropylene Membrane: A Biomimetic Cell Culture Platform with a View to the Improvement of Guided Bone Regeneration.

PurposeDespite the significant advances in modeling of biomechanical aspects of cell microenvironment, it remains a major challenge to precisely mimic the physiological condition of the particular cell niche. Here, the metal-organic frameworks (MOFs) have been introduced as a feasible platform for multifactorial control of cell-substrate interaction, given the wide range of physical and mechanical properties of MOF materials and their structural flexibility.ResultsIn situ crystallization of zeolitic imidazolate framework-8 (ZIF-8) on the polydopamine (PDA)-modified membrane significantly raised surface energy, wettability, roughness, and stiffness of the substrate. This modulation led to an almost twofold increment in the primary attachment of dental pulp stem cells (DPSCs) compare to conventional plastic culture dishes. The findings indicate that polypropylene (PP) membrane modified by PDA/ZIF-8 coating effectively supports the growth and proliferation of DPSCs at a substantial rate. Further analysis also displayed the exaggerated multilineage differentiation of DPSCs with amplified level of autocrine cell fate determination signals, like BSP1, BMP2, PPARG, FABP4, ACAN, and COL2A. Notably, osteogenic markers were dramatically overexpressed (more than 100-folds rather than tissue culture plate) in response to biomechanical characteristics of the ZIF-8 layer.ConclusionHence, surface modification of cell culture platforms with MOF nanostructures proposed as a powerful nanomedical approach for selectively guiding stem cells for tissue regeneration. In particular, PP/PDA/ZIF-8 membrane presented ideal characteristics for using as a barrier membrane for guided bone regeneration (GBR) in periodontal tissue engineering

사람치아 치수줄기세포에서 자가 피브린 매트릭스를 이용한 상아질-펄프 재생

학위논문 (박사) -- 서울대학교 대학원 : 치의학대학원 치의과학과, 2020. 8. 정필훈.Background and Purpose of the Study Xenogeneic serum is widely used as a growth supplement for cell culture medium; however, animal-borne pathogens increase the risk of transmitting infectious agents. Human autologous supplements, including platelet derivatives such as platelet-rich fibrin (PRF), have been assessed in clinical studies as a possible replacement for fetal bovine serum. However, concentrated leukocytes may affect catabolic gene expression in tendons and ligaments, and concentrated red blood cells (RBCs) cause inflammation due to numerous pro-inflammatory interleukins. It was hypothesized that minimizing the number of leukocytes would contribute to the differentiation activity of human dental pulp stem cells (hDPSCs). This study aimed to explore the ability of autologous fibrin (AF) serum prepared after leukocyte removal to induce the differentiation of hDPSCs in vitro and in vivo. Materials and Methods Human DPSCs were obtained from extracted third molars. To characterize the immunophenotype of the hDPSCs, the expression of mesenchymal stem cell-associated surface markers at passage 5 was analyzed by flow cytometry. Next, AF was prepared by high-speed centrifugation and most of the leukocytes were removed. The AF serum was then subjected to three freeze-thaw cycles and used in vitro for the cell culture. hDPSCs were treated with AF serum, and then odontogenic-associated markers were investigated by real-time polymerase chain reaction (PCR). For the in vivo experiments, autologous fibrin matrix (AFM) was fabricated on a plate and hDPSCs cultured on the AFM formed a cell-fibrin complex. Then, the potency of the differentiation induction of the hDPSCs in the cell-fibrin complex was evaluated via real-time PCR and Western blot. The hDPSCs mixed with a cell-fibrin complex were subcutaneously transplanted into nude mice and allowed to grow for 8 weeks. Then, the cell-fibrin complex was examined how induced odontoblastic differentiation and dentin formation of the hDPSCs in the mice by immunohistochemical (IHC) analysis. Results Flow cytometric analysis showed that approximately 90% or more of the hPDLSCs expressed CD13, CD90, and CD146, and 1.58% expressed CD34. The multi-lineage differentiation capacity of hDPSCs in vitro in osteogenic, chondrogenic, and adipogenic medium was observed. Scanning electron microscopy (SEM) images verified that leukocytes were almost absent from the fibrin matrix after high-speed centrifugation. A cytokine array showed that AF serum released sufficient cytokines to induce the differentiation of hDPSCs. AF serum accelerated mineral nodule formation in vitro and increased the expression of odontoblast-associated genes, such as alkaline phosphatase (ALP), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), runt-related transcription factor 2 (Runx2), and vascular endothelial growth factor (VEGF). In addition, the cell-fibrin complex also was observed that it significantly up-regulated the levels of the proteins DMP1, osteopontin (OPN), and DSPP during odontogenic differentiation. Furthermore, IHC staining revealed proteins were expressed after the transplantation of the hDPSCs and the cell-fibrin complex induced the hDPSCs to differentiate into odontoblasts-like cells and to regenerate dentin-pulp tissue in vivo. Conclusions The present study investigated the potential safe methods to expand autologous cells in a risk-free environment, and the cell-fibrin complex scaffold could be generated without the introduction of foreign scaffold materials. The results of experiments confirmed that 1. AF serum prepared after removal of leukocytes to could be used for the proliferation and promotion of hDPSCs odontogenic differentiation under animal serum-free conditions. 2. Human DPSCs cultured on the AFM formed a cell-fibrin complex which became stable and secreted their own matrix to form an autogenous 3D structure. 3. AFM can be effectively used as a carrier for successful regeneration of dentin-pulp complexes, which has been demonstrated by the in vivo subcutaneous transplantation in nude mice. 4. As a clinical implication, AFM possesses good bio-compatibility and cell infiltration and is suitable for dental tissue regeneration in hDPSCs.1. 연구목적 사람치아줄기세포를 배양 시 이종첨가제를 주로 사용하여 왔는데 이는 이종 기원이기 때문에 동물성 병원체 등의 감염원을 지닐 가능성이 있다. 최근 fetal bovine serum (FBS)를 대체하기 위한 임상 연구에서 혈소판 풍부 피브린 (Platelet rich fibrin; PRF)과 같은 혈소판 유도체를 포함한 인간 자가 보충제가 많이 연구되었다. 그러나 PRF는 농축된 백혈구와 적혈구를 함유하고 있으며, 그중 백혈구는 건(tendon)과 인대(ligament)에서 유전자 발현에 영향을 줄 수 있고, 적혈구는 염증반응을 촉진하는 인터루킨을 다량 함유하기 때문에 의도치 않은 염증이 유발될 수 있다고 보고되어왔다. 하여 백혈구 수를 최소화하면 인간치아 치수줄기세포 (human dental pulp stem cells; hDPSCs)의 분화에 기여할 것이라고 가설을 세웠다. 이에 본 연구를 통해 백혈구를 제거한 자가 피브린으로 체외 및 생체 내에서 사람치아 치수줄기세포 의 분화를 유도하는 능력에 대해 알아보고자 한다. 2. 연구방법 발거된 제3대구치의 치수조직에서 사람 치수줄기세포를 추출하였으며, 이를 대상으로 줄기세포 특성을 조사하였다. 사람 혈액을 고속 원심 분리하여 자가 피브린을 만들었고 그 피브린 추출물로 in vitro 실험을 위한 세포 배양에 사용되었으며, 또한 자가 피브린 매트릭스를 사용하여 in vivo 실험을 시행하였다. 우선, 혈액을 원심 분리하여 얻은 자가 피브린 매트릭스에서 백혈구가 제거되었는지 확인하기 위하여 조직학적 분석 및 주사전자현미경(scanning electron microscopy; SEM) 촬영을 통해 확인하였고, 사람 치수줄기세포에 자가 피브린 추출물을 처리한 뒤 qRT-PCR (quantitative real-time polymerase chain reaction)을 통해 치성 관련 유전자 발현을 조사하였다. 그리고 치수줄기세포를 자가 피브린 매트릭스 위에서 배양하여 줄기세포-피브린 매트릭스 혼합체를 얻어 그 효능을 Western blot을 통해 평가하였다. in vivo 환경에서의 영향을 확인하기 위해 누드마우스를 대상으로, 실험군으로 치수줄기세포와 자가 피브린 매트릭스 혼합체를 한 쪽 피하에 이식하였고, 대조군으로 자가 피브린 매트릭스를 실험군 반대편에 이식하였다. 8주 후 이식한 조직을 채취하여 상아질모세포(odontoblast) 분화와 상아질모세포에 의한 상아질 생성 및 특정된 단백질 발현을 immunohistochemistry로 비교 확인하였다. 3. 연구결과 유세포 분석 결과 치수줄기세포 표지인자인 CD13, CD90, CD146이 발현 되었으며, 분화를 통해 다중 분화 능력이 있는 줄기세포의 특징을 가지고 있음을 확인하였다. 조직학적 분석과 SEM으로 확인한 결과 자가 피브린 매트릭스에서 백혈구는 거의 확인되지 않았다. In vitro에서 자가 피브린 추출물은 경조직 분화 마커인 alkaline phosphatase (ALP), bone sialoprotein (BSP), dentin matrix protein 1 (DMP1), dentin sialophosphoprotein (DSPP), runt-related transcription factor 2 (Runx2), vascular endothelial growth factor (VEGF)의 발현이 증가한 것을 확인하였다. 또한 줄기세포-매트릭스 혼합체는 상아질모세포의 분화과정에서 DMP1, osteopontin (OPN) 및 DSPP의 단백질 발현을 유의하게 촉진함을 확인할 수 있었다. 누드마우스의 피하에 이식한 매식체의 조직학적 분석결과 대조군에 비해 치수줄기세포와 자가 피브린 매트릭스 혼합체의 이식군에서 상아질 특정 단백질이 더욱 강하게 염색되었으며, 상아질-펄프 조직이 재생되어 있음을 확인하였다. 4. 결론 이 연구는 무위험 환경에서 자가 세포를 확장시키는 잠재적인 안전한 방법을 조사했으며, 세포 스캐폴드 복합체는 외인성 물질의 도입없이 치아 조직이 생성 될 수 있다. 1. 자가 피브린 추출물은 백혈구를 제거한후 제조되었으며 동물 무 혈청 조건하에 치아줄기세포의 증식과 분화에 유용하게 이용가능하다. 2. 자가 피브린 매트릭스에서 배양된 치아줄기세포는 세포-피브린 복합체를 형성하였으며, 이는 안정적이고 3차원적인 자가 매트릭스 구조를 형성하였다. 3. 자가 피브린 매트릭스는 생체 내 누드마우스 피하에 이식하여 상아질-펄프 복합체의 성공적인 재생에 우수한 운반체로서 유용하게 이용할 수 있음이 입증되었다. 4. 임상적 의미에서 자가 피브린 매트릭스는 우수한 생체 적합성 및 세포 침윤을 보유하며 치아줄기세포에서 치아조직 재생에 유용하다는 것을 알 수 있다.Ⅰ. INTRODUCT 1 Ⅱ. MATERIALS AND METHODS 5 1. Samples 5 2. Fabrication of AF and AF serum 5 3. Histological analysis of AF and PRF 6 4. Scanning electron microscopy (SEM) analysis 7 5. Human cytokine antibody array 8 6. Primary cell culture 9 7. Proliferation assay 10 8. Flow cytometry analysis 10 9. Multi-differentiation of hDPSCs 11 10. Gene expression of the hDPSCs 12 11. Fabrication of autologous fibrin matrix (AFM) and dental pulp stem cells culture on the AFM (cell-fibrin complex) 13 12. The invasion of the hDPSCs into the AFM 14 13. Immunofluorescence analysis of cell adhesion 15 14. Evaluation of the differentiation of hDPSCs on the AFM by quantitative real-time PCR 15 15. Western blot analysis 16 16. Transfection of AFM into a rat calvarial defect 17 17. Micro-computed tomography imaging analysis 18 18. Preparation of human dentin matrix 18 19. Transplantation 19 20. Histological analysis 20 21. IHC analysis 21 22. Statistical analysis 22 Ⅲ. RESULTS 1. Characterization of AF 23 2. Detection of cytokines in the AF serum 24 3. Characterization of hDPSCs in the AF serum 25 4. Effects of the AF serum on the hDPSCs in vitro 26 5. Optimal thickness of the AFM for the hDPSCs culture 27 6. Infiltration and morphological observation of the hDPSCs on AFM 28 7. The odontogenic influences of hDPSCs on the AFM 28 8. Effects of AFM on the rat calvarial defect model 29 9. Histological analysis of human dentin matrix surface in vivo 30 Ⅳ. DISCUSSION 32 Ⅴ. CONCLUSIONS 43 Ⅵ. REFERENCES 44 Ⅶ. FIGURES AND TABLE 54 Ⅷ. ABSTRACT IN KOREAN 77 LIST OF FIGURES Figure 1. Fabrication of an AF and AF serum 54 Figure 2. AF compared with PRF 55 Figure 3. SEM evaluation of the AF 56 Figure 4. Evaluation of a human cytokine/chemokine array for AF serum 57 Figure 5. Characterization of hDPSCs at passage 5 by flow cytometry assay 58 Figure 6. Characterization of hDPSCs 59 Figure 7. Osteogenic differentiation of hDPSCs in vitro 60 Figure 8. Morphology of hDPSCs cultured in AF serum compared with FBS 61 Figure 9. Effects of AF serum on the proliferation of hDPSCs compared with 10% FBS in vitro 62 Figure 10. Effects of AF serum on the odontogenic influence of hDPSCs in vitro 63 Figure 11. Images of hDPSCs cultured on the AFM (cell-fibrin complex) 64 Figure 12. Optimal AFM thickness for hDPSCs culture 65 Figure 13. Infiltration of DPSCs on AFM 66 Figure 14. Morphological observation of hDPSCs on AFM 67 Figure 15. Evaluation of the odontogenic differentiation of hDPSCs cultured on the AFM 68 Figure 16. Investigation of odontogenic-specific protein expression by Western blot assay 69 Figure 17. Effects of AFM on the rat calvarial defect model 70 Figure 18. Morphology of the human dentin matrix 71 Figure 19. Image of individual components of the transplantation composite and schematic of the final construct 72 Figure 20. Effects on the AFM surface in vivo 73 Figure 21. Immunohistochemical analysis showing the effect of the hDPSCs cultured on the AFM in vivo (low magnification) 74 Figure 22. Immunohistochemical analysis showing the effect of the hDPSCs cultured on the AFM in vivo (high magnification) 75Docto

Dental Mesenchymal Stem Cell-Based Translational Regenerative Dentistry: From Artificial to Biological Replacement

Dentistry is a continuously changing field that has witnessed much advancement in the past century. Prosthodontics is that branch of dentistry that deals with replacing missing teeth using either fixed or removable appliances in an attempt to simulate natural tooth function. Although such “replacement therapies” appear to be easy and economic they fall short of ever coming close to their natural counterparts. Complications that arise often lead to failures and frequent repairs of such devices which seldom allow true physiological function of dental and oral-maxillofacial tissues. Such factors can critically affect the quality of life of an individual. The market for dental implants is continuously growing with huge economic revenues. Unfortunately, such treatments are again associated with frequent problems such as peri-implantitis resulting in an eventual loss or replacement of implants. This is particularly influential for patients having co-morbid diseases such as diabetes or osteoporosis and in association with smoking and other conditions that undoubtedly affect the final treatment outcome. The advent of tissue engineering and regenerative medicine therapies along with the enormous strides taken in their associated interdisciplinary fields such as stem cell therapy, biomaterial development, and others may open arenas to enhancing tissue regeneration via designing and construction of patient-specific biological and/or biomimetic substitutes. This review will overview current strategies in regenerative dentistry while overviewing key roles of dental mesenchymal stem cells particularly those of the dental pulp, until paving the way to precision/translational regenerative medicine therapies for future clinical use