The evolving regulatory system of advanced therapy medicinal products in China: a documentary analysis using the World Health Organization Global Benchmarking Tool standards.

Advanced therapy medicinal products (ATMPs) are rapidly evolving to offer new treatment options. The scientific, technical, and clinical complexities subject drug regulatory authorizes to regulatory challenges. To advance the regulatory capacity for ATMPs, the National Medical Products Administration in China made changes to the drug regulatory system and developed regulatory science with the goal of addressing patient needs and encouraging innovation. This study aimed to systematically identify the regulatory evidence on ATMPs in China under the guidance of an overarching framework from the World Health Organization Global Benchmarking Tool. It was found that China's administrative authorities at all levels have issued a number of policy documents to promote the development of ATMPs, covering biopharmaceutical products research and development (n = 14), biopharmaceutical industry development (n = 9), high-quality development of medical institutions (n = 1), specific development plans/projects (n = 6) and specific regional development (n = 4). The legal and regulatory framework of ATMPs in China has been established and is subject to continuous adjustment in various aspects including regulations (n = 3), departmental rules or administrative normative documents (n = 22), and technical guidance (n = 15). As the regulatory reform continues, the drug review processes have been revised, and various technical standards have been launched, which aim to establish a regulatory approach that oversees the full life-cycle development of ATMPs in the country. The limited number of investigational new drug applications and approved ATMPs suggests a lag remains between the translation of advanced therapeutic technologies into clinically available medical products. To accelerate the translational research of ATMP in countries such as China, developing and adopting real-world evidence generated from clinical use in designated healthcare facilities to support scientific decision-making in ATMP regulation is warranted. The enhancement of regulatory capacity building and multi-stakeholder collaborations should also be encouraged to facilitate the timely evaluation of promising ATMPs to meet more patient needs.

Is there a role for traditional and complementary medicines in managing chronic fatigue? a systematic review of randomized controlled trials.

Introduction: Chronic fatigue syndrome (CFS) is an increasingly common condition that is challenging to treat due to unclear etiology and a lack of consensus on clinical diagnosis and treatment guidance. Many affected people resorted to using traditional and complementary medicines (T&CMs). However, the evidence for T&CMs for CFS has been inconclusive and continues to evolve. The study aims to identify, summarize and assess the most recent evidence on the efficacy and safety of T&CMs for CFS. Methods: Randomized controlled trials (RCTs) investigating T&CMs for CFS published in English of Chinese between 1 January 2013 and 31 December 2022 were searched from 7 databases. RCTs comparing T&CMs with no treatment, placebo, or pharmacological medicine were included, irrespective of language or blinding. The Consolidated Standards of Reporting Trials Statement extensions for Chinese herbal medicine Formulas (CONSORT-CHM) and the Cochrane Collaboration's Risk of Bias tool were used to evaluate the quality and risk of bias of included studies. Results: A total of 62 RCTs investigating 43 types of T&CMs and involving 5,231 participants with CFS were included in this review. The primary outcome measures mainly included the scoring of fatigue symptoms using the validated tool Fatigue Scale-14 (FS-14) or the TCM syndrome score. The main interventions showing overall efficacy were Chaihu Guizhi Decoction and Buzhong Yiqi combined with Xiao Chaihu Decoction, and 148 ingredients were identified, including Astragali Radix, Glycyrrhizae Radix et Rhizoma, Atractylodis Macrocephalae Rhizoma, and Bupleuri Radix. The most significant effect was the improvement of fatigue, followed by TCM-diagnosed symptoms and other psychological conditions. No serious adverse effect had been reported. However, the quality of the RCTs included RCTs were found to be suboptimal, and the risk of bias remained uncertain. Conclusion: Some evidence from RCTs supported the efficacy and safety of T&CM in CFS. However, given the methodological and quality heterogenicity of the included studies, the recommendations of T&CMs in treating CFS remain inconclusive. To develop better quality evidence about T&CMs for CFS, future studies should employ more objective diagnosis standards and outcome measurements, larger sample size, and better bias control, and ensure the compliance with the corresponding reporting guidelines. Systematic Review Registration: https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42022362268, identifier CRD42022362268.

Efficacy and safety of traditional Chinese medicine for cancer-related fatigue: a systematic literature review of randomized controlled trials.

BACKGROUND: Cancer-related fatigue (CRF) is an extremely common and long-term condition that affects the physical and mental health of oncology patients. While the treatment for CRF with western medicine and non-pharmacological therapy remains uncertain and challenging, traditional Chinese medicine (TCM) has become a trending option for the patients. Based on the findings from randomized controlled trials (RCTs), this study aims to identify and evaluate the evidence about the efficacy and safety of TCM for CRF. METHODS: A systematic literature search was conducted according to the PRISMA literature research guidelines. Seven electronic databases including PubMed, the Cochrane Library, Embase, Web of Science, Scopus, China National Knowledge Infrastructure (CNKI) and Wanfang database were searched to identify RCTs which investigated TCM in the treatment of CRF published since inception to December 2022. RCTs comparing TCM with no treatment, placebo, or pharmacological interventions were considered eligible for this review. The Consolidated Standards of Reporting Trials Statement extensions for Chinese herbal medicine Formulas (CONSORT-CHM) and the Cochrane Collaboration's Risk of Bias tool were used in this review to evaluate the quality and the risk of bias of all included trials. RESULTS: A total of 82 RCTs were included in this review, regardless of whether they were published in English or Chinese. After data extraction and results evaluation, 78 trials demonstrated overall efficacy in using TCM for CRF patients compared with the control group, in which 33 trials showed that the efficacy rate was statistically significant (p < 0.05 or p < 0.01). TCM was also shown to be beneficial in improving the scores of relevant scales (e.g., PFS, QoL, TCM syndrome score, other fatigue scales etc.) or physical tests indicators (e.g., cytokines, blood test etc.). The most common herbs found in Chinese medicine were Astragali Radix, Ginseng Radix and Codonopsis Radix. Some TCM products, such as Kangai Injection, Buzhong Yiqi Decoction and Shenqi Fuzheng Injection could provide a reference for medication in this review. A range of non-serious, reversible adverse effects associated with the use of TCM was also reported. However, the result of evaluation showed that none of the trials fully met all the CONSORT-CHM criteria, the quality of included trials was generally poor and the risk of bias was mostly uncertain. CONCLUSION: There is some evidence supporting the efficacy and safety of TCM in managing CRF in this systematic review. However, no clear conclusion can be made due to the inadequate reporting of efficacy and adverse reactions. In view of some concerns about the existing evidence after the evaluation, it is essential to standardize the comprehensive identification and efficacy measurement standards, improve the quality of RCTs and conduct more multicomponent therapies to provide an updated reference for CRF patients medication in the future. The protocol of this systematic review has been registered on PROSPERO (CRD42023413625). [ https://www.crd.york.ac.uk/prospero/display_record.php?ID=CRD42023413625 ].

Systematic analysis of randomised controlled trials of Chinese herb medicine for non-alcoholic steatohepatitis (NASH): implications for future drug development and trial design.

BACKGROUND: Non-alcoholic steatohepatitis (NASH) is a liver disease currently lacking an approved therapy, resulting in significant clinical demand. Traditional Chinese medicines (TCMs) have been commonly used to manage NASH. This study aimed to systematically analyse the randomised controlled trials (RCTs) using TCMs for NASH management. METHODS: A systematic literature review was performed by following PRISMA guidelines 2020 in six electronic databases: PubMed, Web of Science, Scopus, Embase, the Cochrane Library, and China National Knowledge Infrastructure, from inception until August 2022. RCTs using TCMs for NASH were included in the analysis, irrespective of language or blinding. RESULTS: 112 RCTs were included in this review, with 10,573 NASH participants. 108 RCTs were conducted in China, and 4 RCTs were in other countries. Herbal medicine decoction was the major dosage form used for treating NASH (82/112). 11 TCMs products have been approved for NASH treatment (8 in China, 2 in Iran, and 1 in Japan). Classic prescriptions, such as "Huang Lian Jie Du decoction", "Yin Chen Hao decoction", and "Yi Guan Jian" were used in some studies. The TCMs treatment of NASH involved the use of 199 different plants, with the top 5 herbs being Salviae Miltiorrhizae Radix Et Rhizoma, Alismatis Rhizoma, Bupleuri Radix, Poria, and Curcumae Radix. "Salviae Miltiorrhizae Radix Et Rhizoma + Bupleuri Radix/Alismatis Rhizoma" were the mostly common drug-pair in the herbs network analysis. Nowadays, "Bupleuri Radix/Alismatis Rhizoma + Atractylodis Macrocephalae Rhizoma" are increasingly applied in herbal formulas for NASH. Based on the PICOS principles, the included studies varied in terms of the population, intervention, comparator, outcomes, and study design. However, some studies reported unstandardised results and failed to report diagnostic standards, inclusion or exclusion criteria, or sufficient patient information. CONCLUSION: Adopting Chinese classic prescriptions or drug-pair may provide a basis for developing new drugs of NASH management. Further research is needed to refine the clinical trial design and obtain more convincing evidence for using TCMs to treat NASH.

Application of implementation science framework to develop and adopt regulatory science in different national regulatory authorities.

Introduction: The purpose of developing and adopting regulatory science (RS) for drug regulatory authorities (DRAs) is to enhance regulatory capacity by advancing the scientific approach for the evaluation of health-related products. While many DRAs around the world advocate the concept of RS, the implementation approaches of RS vary according to local needs and have not been systemically examined. This study aimed to systematically identify the evidence about how RS was developed, adopted, and advanced by the selected DRAs, and analyzed and compared the implementation experiences of RS development under the guidance of an implementation science framework. Methods: Documentary analysis of government documents and a scoping literature review were conducted, and data analysis was performed under the guidance of the PRECEDE-PROCEED Model (PPM). DRAs in the United States, the European Union, Japan, and China had officially launched RS initiatives and were therefore selected as the target countries in this study. Results: There is no common consensus on the definition of RS among the DRAs. However, these DRAs shared the same goal of developing and adopting RS, which was used to develop new tools, standards, and guidelines that could improve the effectiveness and efficiency of the risk and benefit assessment of the regulated products. Each DRA had decided its own priority areas for RS development and thus set specific objectives that might be technology-based (e.g., toxicology and clinical evaluation), process-based (e.g., partnership with healthcare systems and high-quality review/consultation services), or product-based (e.g., drug-device combination products and innovative emerging technologies). To advance RS, considerable resources had been allocated for staff training, advancing information technology and laboratory infrastructure, and funding research projects. DRAs also took multifaceted approaches to expand scientific collaborations through public-private partnerships, research funding mechanisms, and innovation networks. Cross-DRA communications were also reinforced through horizon scanning systems and consortiums to better inform and assist the regulatory decision-making process. The output measurements might be scientific publications, funded projects, DRAs interactions, and evaluation methods and guidelines. Improved regulatory efficiency and transparency leading to benefits to public health, patient outcomes, and translation of drug research and development as the key primary outcomes of RS development were anticipated but not yet clearly defined. Conclusion: The application of the implementation science framework is useful for conceptualizing and planning the development and adoption of RS for evidence-based regulatory decision-making. Continuous commitment to the RS development and regular review of the RS goals by the decision-makers are important for DRAs to meet the ever-changing scientific challenges in their regulatory decision-making process.

Benchmarking Drug Regulatory Systems for Capacity Building: An Integrative Review of Tools, Practice, and Recommendations.

BACKGROUND: Benchmarking has been increasingly used on drug regulatory systems to achieve sustainable pharmaceutical system strengthening. This study aimed to identify the scope, tools and benefits of benchmarking regulatory capacities and the most recent development in such phenomenon. Method: This study employed an integrative and critical review of the literature and documents on benchmarking drug regulatory capacities identified from 6 databases and 5 websites of related organizations and government agencies in compliance with the Preferred Reporting Items for Systematic Review (PRISMA) guidelines. RESULTS: Forty-three studies and 6 documents about regulatory benchmarking published between 2005 and 2022 were included in this review. Five benchmarking assessment tools or programmes recommended or adopted by international organizations or government agencies had been identified, which collectively covered 12 major regulatory functions (4 at system level and 8 at operational level) involving 9 indicator categories and 382 sub-indicators. Benchmarking drug regulatory systems was reportedly employed at national, regional and international levels for either internal assessment (mostly on regulatory system establishment, drug review process and post marketing surveillance) or external evaluation (mostly on regulatory standards, drug review process and pharmacovigilance systems) to assess current status, monitor performance, determine major challenges and inform actions for capacity building. Priority of actions in areas such as regulatory process, resources allocation, cooperation and communication, and stakeholder engagement have been suggested for strengthening drug regulatory systems. Nevertheless, the evidence about benchmarking in optimizing regulatory capacities remained underreported. CONCLUSION: This integrative review depicted a framework for decision-makers about why and how benchmarking drug regulatory systems should be undertaken. For effective benchmarking, well-informed decisions about the goals, the scope, the choice of reference points and benchmarking tools are essential to guide the implementation strategies. Further studies about the positive effects of regulatory benchmarking are warranted to engage continuous commitment to the practice.

Integrating Real-World Evidence in the Regulatory Decision-Making Process: A Systematic Analysis of Experiences in the US, EU, and China Using a Logic Model.

Real world evidence (RWE) and real-world data (RWD) are drawing ever-increasing attention in the pharmaceutical industry and drug regulatory authorities (DRAs) all over the world due to their paramount role in supporting drug development and regulatory decision making. However, there is little systematic documentary analysis about how RWE was integrated for the use by the DRAs in evaluating new treatment approaches and monitoring post-market safety. This study aimed to analyze and discuss the integration of RWE into regulatory decision-making process from the perspective of DRAs. Different development strategies to develop and adopt RWE by the DRAs in the US, Europe, and China were reviewed and compared, and the challenges encountered were discussed. It was found that different strategies on development of RWE were applied by FDA, EMA, and NMPA. The extent to which RWE was adopted in China was relatively limited compared to that in the US and EU, which was highly related to the national pharmaceutical environment and development stages. A better understanding of the overall goals, inputs, activities, outputs, and outcomes in developing RWE will help inform actions to harness RWD and leverage RWE for better health care decisions.

Applying regulatory science in traditional chinese medicines for improving public safety and facilitating innovation in China: a scoping review and regulatory implications.

BACKGROUND: The National Medical Products Administration (NMPA) in China has set to advance the regulatory capacity of traditional Chinese medicines (TCMs) with the adoption of regulatory science (RS). However, the priority of actions at the interface of RS and TCMs were yet to be defined. This research aims to identify the priority areas and summarize core actions for advancing RS for traditional medicines in China. METHODS: A mixed approach of documentary analysis of government policies, regulations and official information about TCMs regulation in China, and a scoping review of literature using 4 databases (PubMed, ScienceDirect, Scopus and CNKI) on major concerns in TCMs regulation was employed. RESULTS: Ten priority areas in the development of TCM-related regulatory science in China have been identified, including: (1) modernizing the regulatory system with a holistic approach; (2) advancing the methodology for the quality control of TCMs; (3) fostering the control mechanism of TCMs manufacturing process; (4) improving clinical evaluation of TCMs and leveraging real world data; (5) re-evaluation of TCMs injection; (6) developing evaluation standards for classic TCMs formula; (7) harnessing diverse data to improve pharmacovigilance of TCMs; (8) evaluating the value of integrative medicine in clinical practice with scientific research; (9) advancing the regulatory capacity to encourage innovation in TCMs; and (10) advancing a vision of collaboration for RS development in TCMs. CONCLUSIONS: RS for TCMs in China encompasses revolution of operational procedures, advancement in science and technology, and cross-disciplinary collaborations. Such experiences could be integrated in the communications among drug regulatory authorities to promote standardized and scientific regulation of traditional medicines.

Efficacy and safety of traditional Chinese medicines for non-alcoholic fatty liver disease: a systematic literature review of randomized controlled trials.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is a common liver disease that may progress into, in the absence of proper treatment, severe liver damage. While the optimal pharmacotherapy for NAFLD remains uncertain and the adherence to lifestyle interventions is challenging, the use of herbal medicines such as traditional Chinese medicines (TCMs) to manage the condition is common. The evidence about TCMs in the management of NAFLD is continuously developing through randomized controlled trials (RCTs). This study aims to identify and evaluate the emerging evidence about the efficacy and safety of TCMs for NAFLD. METHODS: A systematic literature search was conducted to identify RCTs which investigated TCMs in the management of NAFLD published in 6 electronic databases including PubMed, the Cochrane Library, EMBASE, Web of Science, Scopus and China National Knowledge Infrastructure since inception to September 2020. RCTs comparing TCMs with no treatment, placebo, non-pharmacological and/or pharmacological interventions were included irrespective of language or blinding. The quality of reporting was evaluated using the Consolidated Standards of Reporting Trials Statement extensions for Chinese herbal medicine Formulas (CONSORT-CHM). Risk-of-bias for each study was assessed using the Cochrane risk of bias tool. RESULTS: A total of 53 RCTs involving 5997 participants with NAFLD were included in this review. Each included RCT tested a different TCMs giving a total of 53 TCMs identified in this study. Based on the evaluation of the RCT results, TCMs might have various beneficial effects such as improving TCM syndrome score, liver function, and body lipid profile. A range of non-serious, reversible adverse effects associated with the use of TCMs were also reported. However, no conclusion about the efficacy and safety of TCMs in NAFLD can be made. The quality of reporting was generally poor and the risks of bias was mostly uncertain in all trials. CONCLUSIONS: There is some evidence from RCTs that supported the effectiveness and safety of TCMs for NAFLD. However, no conclusive recommendations can be made due to the questionable quality of the RCTs. Improvement in the RCT protocol, the use of a larger sample size, a setting of multicenter, and a more focused approach in selecting TCMs are recommended for developing high quality evidence about the use of TCMs in managing NAFLD.

[Dynamic change and prediction of vegetation cover in Shenzhen, China from 2000 to 2018]. / 2000­2018年深圳市植被覆盖动态变化与预测.

With landsat-series multi-temporal image data, percentage of vegetation cover (PVC) was estimated by pixel dichotomy. The linear regression analysis and center of gravity migration methods were used to explore the characteristics of the spatiotemporal changes of vegetation cover in Shenzhen from 2000 to 2018. The CA-Markov model was combined to predict future land cover in Shenzhen. The results showed that the PVC in Shenzhen demonstrated obvious regional differentiation characteristics from 2000 to 2018. The eastern region occupied larger proportion than the wes-tern part, while the southern region was larger than the north part. This feature exhibited good consistency with regional topographic effect. The spatial migration characteristic of the center of gravity of PVC was from northwest to southeast, and then from southeast to northwest, with a migration rate of 551.2 m·a-1. This process was closely related to urbanization in Shenzhen. The PVC in Shen-zhen tended to be generally improved from 2000-2018, with a improvement rate of 0.005·a-1. The percentage of significantly improved and degraded PVC area was 30.8% and 12.8%, respectively. The CA-Markov method was used to predict the land cover/use pattern of Shenzhen in 2024 under two scenarios, theoretical scenario and natural scenario. There was no significant difference in proportion of the area of the land cover/use patterns obtained by the two kinds of prediction method, with the difference threshold being 0-1.2%. Compared with the data before 2018, the proportion of arbor forests and arable land converted into construction land in Shenzhen would be significantly reduced in 2024, whereas the contradiction between supply and demand would be still tense.

An evaluation of randomized controlled trials on nutraceuticals containing traditional Chinese medicines for diabetes management: a systematic review.

BACKGROUND: Nutraceuticals containing traditional Chinese medicine (TCM) are promoted for use in the management of diabetes. The evidence to support such use is largely unknown. This study aimed to summarise and evaluate the literature reporting the results of randomized controlled trials (RCTs) investigating the effects of nutraceuticals in people living with diabetes. METHODS: Literature from four electronic databases (PubMed, Scopus, CINAHL and Web of Science) was searched following PRISMA guidelines to yield RCT publications on nutraceutical for diabetes management published since 2009. The quality of reporting was assessed using the CONSORT 2010 checklist statement. Risk-of-bias for each study was assessed using the Cochrane risk of bias tool. RESULTS: Out of 1978 records identified in the initial search, 24 randomized, double/triple-blinded, controlled trials that investigated the effect of nutraceuticals covering 17 different TCM herbs for diabetes management were selected. Participants included people who were diabetic (n = 16), pre-diabetic (n = 3) or predisposed to diabetes (n = 5). Sample sizes ranged between 23 and 117 for 2 arms, or 99-165 for 3 arms. Comparisons were made against placebo (n = 22), conventional medicine (n = 1), or regular diet (n = 1) for a duration between 4 and 24 weeks. All but one study tested the effect on fasting blood glucose levels (n = 23) or glycated haemoglobin levels (n = 18), and/or postprandial 2-h blood glucose levels (n = 4) as the primary outcomes. Nineteen studies reported some statistically significant reductions in the respective measures while 5 studies showed no effect on primary or secondary outcomes. None of the included studies met all the criteria for the CONSORT guidelines. Incomplete reporting about randomization and blinding, and a lack of ancillary analyses to explore other influential factors and potential harms associated with the use were repeatedly noted. Based on the Cochrane risk-of-bias tool, 19 studies were deemed to have a high risk of bias mainly attributed to sponsor bias. CONCLUSIONS: There is some evidence to suggest positive clinical outcomes in response to the administration of a range of nutraceuticals containing TCM in the management of diabetes. However, these results must be interpreted with caution due to the overall low quality of the trials.

[Effects of tooth germ microenvironment in vitro on the differentiation of dental pulp stem cell and ectoblast mesenchyme stem cell].

OBJECTIVE: To observe the differential ability of dental pulp stem cell (DPSC) and ectoblast mesenchyme stem cell (EMSC) that were cultured with tooth germ cell (TGC) as the tooth germ microenvironment. METHODS: The TGC of 4-day old rat was used as the tooth germ microenvironment. The BrdU marked and determined DPSC and EMSC were cultured with the TGC respectively. The expression of cell surface antigen dentin sialoprotein (DSP) and alkaline phosphatase (ALP) activity were determined with double marker immunofluorescence. The differential ability of DPSC and EMSC were determined by the immunohistochemistry and image analysis in the tooth germ microenvironment. RESULTS: The transformation efficiency of DSP positive cell in the EMSC co-culture group was higher than that in the DPSC co-culture group (P < 0.05). The transformation efficiency in the co-culture groups was higher than that in the non co-culture group after 7 days by the image analysis of immunohistochemistry (P < 0.05). The ALP activity in the co-culture groups increased after 3 and 7 days. The ALP activity in the EMSC co-culture group was higher than that in the DPSC co-culture group. CONCLUSION: DPSC and EMSC cultured with TGC as the tooth germ microenvironment can be induced to differentiate into odontoblast. The ability of EMSC is higher than that of DPSC.

Odontoblast-like cell differentiation and dentin formation induced with TGF-ß1.

OBJECTIVE: To investigate the inductive potential of scaffold material combing with transforming growth factor-ß1 (TGF-ß1), and to induce odontoblast differentiation and dentin formation from dental pulp cells both in vitro and in vivo. METHODS: Primarily cultured dental pulp cells were used for MTT, ALP activity assay and Alizarin red staining in the presence of TGF-ß1. Pelleted cells were put on the filters combining with or not with TGF-ß1 and cultured in vitro or in vivo. The in vitro and in vivo cell response and tissue formation were analysed with Haematoxylin-Eosin (HE), transmission electron microscopy (TEM) and immunohistochemical staining. RESULTS: TGF-ß1 increased the mineralization and ALP activity of dental pulp cells as revealed by Alizarin red staining and ALP activity assay. After in vitro culture for 7 days, cells polarized in the TGF-ß1 group and expressed dentin sialoprotein (DSP), osteopontin (OPN) and type I collagen (Col I). After in vivo transplantation for 7 days, columnar odontoblast formed on the surface of filter in experimental group, and tubular dentin expressing DSP formed after 3 months transplantation. CONCLUSION: It was concluded that TGF-ß1 combining with transfilter could induce odontoblast differentiation and dentin formation. Our results implied that suitable substrate for the progenitors of odontoblast to anchor on and inductive signals to initiate the differentiation of odontoblast should be taken into consideration when designing scaffold material for inducing dentin tissue engineering.

Differentiation potential of STRO-1+ dental pulp stem cells changes during cell passaging.

BACKGROUND: Dental pulp stem cells (DPSCs) can be driven into odontoblast, osteoblast, and chondrocyte lineages in different inductive media. However, the differentiation potential of naive DPSCs after serial passaging in the routine culture system has not been fully elucidated. RESULTS: DPSCs were isolated from human/rat dental pulps by the magnetic activated cell sorting based on STRO-1 expression, cultured and passaged in the conventional culture media. The biological features of STRO-1+ DPSCs at the 1st and 9th passages were investigated. During the long-term passage, the proliferation ability of human STRO-1+ DPSCs was downregulated as indicated by the growth kinetics. When compared with STRO-1+ DPSCs at the 1st passage (DPSC-P1), the expression of mature osteoblast-specific genes/proteins (alkaline phosphatase, bone sialoprotein, osterix, and osteopontin), odontoblast-specific gene/protein (dentin sialophosphoprotein and dentin sialoprotein), and chondrocyte-specific gene/protein (type II collagen) was significantly upregulated in human STRO-1+ DPSCs at the 9th passage (DPSC-P9). Furthermore, human DPSC-P9 cells in the mineralization-inducing media presented higher levels of alkaline phosphatase at day 3 and day 7 respectively, and produced more mineralized matrix than DPSC-P9 cells at day 14. In vivo transplantation results showed that rat DPSC-P1 cell pellets developed into dentin, bone and cartilage structures respectively, while DPSC-P9 cells can only generate bone tissues. CONCLUSIONS: These findings suggest that STRO-1+ DPSCs consist of several interrelated subpopulations which can spontaneously differentiate into odontoblasts, osteoblasts, and chondrocytes. The differentiation capacity of these DPSCs changes during cell passaging, and DPSCs at the 9th passage restrict their differentiation potential to the osteoblast lineage in vivo.

Odontogenic potential of mesenchymal cells from hair follicle dermal papilla.

Dental pulp stem cells from teeth can be used for tooth regeneration. Although nondental stem cells derived from bone marrow can differentiate into odontoblast-like cells when recombined with embryonic oral epithelium, these cells can lose their ability to differentiate after an extended number of cell culture passages. There has been limited research to identify stem cells from other tissue sources to regenerate teeth. As another candidate source for mesenchymal stem cells, hair follicle has obtained much more attention recently because of its easy accessibility. In this study, cultured vibrissae follicle dermal papilla mesenchymal cells (FDPMCs) from adult C57BL/6 GFP mice can differentiate into adipocytes and osteoblasts in vitro. Moreover, in the inductive microenvironment generated by apical bud and dental mesenchyme from 7-day-old C57 mice, FDPMCs in vitro demonstrated odontogenic potential, as indicated by the morphological transformation, cell-cycle change and expression of tooth-specific markers. Under the same microenvironment, FDPMCs were incubated in vivo for 3 weeks. Coexpression of GFP and DSP proteins in the odontoblast layer was detected in the recovered implants, suggesting that GFP(+) FDPMCs can function as odontoblasts in vivo. Together, our data indicate for the first time that whisker FDPMCs from adult mice can differentiate to odontoblast-like cells.

Cementum and periodontal ligament-like tissue formation induced using bioengineered dentin.

Stem cell-mediated root regeneration offers opportunities to regenerate a bio-root and its associated periodontal tissues to restore tooth loss. Periodontal ligament (PDL) and cementum complex and dentin pulp complex have been tissue engineered using human dental pulp stem cells and PDL stem cells, respectively. The aim of this study was to explore whether dentin formation could be induced using an inductive substrate and whether bioengineered dentin could induce cementum and PDL formation. First, dentin was bioengineered from tooth papillae of Sprague-Dawley (SD) rats with an inductive substrate, and its phenotype was characterized; then primarily cultured human PDL cells were seeded on the surface of dentin and transplanted under the skin of immunocompromised mice. Histological, immunohistochemical, and scanning electronic microscopy examinations results showed that bioengineered dentin could induce cementogenesis and PDL formation, and condense PDL arranged perpendicularly on the dentin surface via a layer of cementum-like tissue. The results indicated that tissue-engineered dentin could be induced using an inductive substrate and could be used as a further substrate for cementum and PDL tissue engineering.

Current approaches and challenges in making a bio-tooth.

Tooth loss adversely affects not only mouth functions but also the esthetics of one's face. To repair these defects, current treatment methods mainly depend on nonbiological materials or artificial implants that also can, sometimes, reduce the quality of life because of their limited physiological function, or elicit an immunological rejection. Theoretically, a biological tooth (bio-tooth) that is made from the patient's own cells and grows in its intended location should be the best choice for treating tooth loss, although such bioengineered teeth have been nothing more than a dream for many centuries. Recently, significant advances in the fields of tissue engineering, stem cell biology, developmental biology, molecular genetics, and bionics have brought us close to the realization of a bio-tooth. However, issues involving in the reconstruction of a bio-tooth regarding the shape determination, size control, availability of dental epithelium, directional growth and eruption, and graft rejection in the jaws remain to be resolved. Here, this review outlines the current approaches toward the tooth regeneration, and focuses on several key challenges that must be met in the making of a bio-tooth.

Epithelial-mesenchymal cell ratios can determine the crown morphogenesis of dental pulp stem cells.

Although dental pulp stem cells (DPSC) have been isolated from adult dental pulp tissues, knowledge on how to use them to make teeth lags behind. To date, little is known about the effects of epithelial-mesenchymal cell ratios on the bioengineered odontogenesis mediated by DPSCs. In this study, we investigated the effects of apical bud cells (ABC) from dental epithelial stem cell niche of rat incisors on the differentiation and morphogenesis of molar DPSCs at different proportions (DPSC/ABC cell ratios=1:10, 1:3, 1:1, 3:1, 10:1, respectively). In vitro mixed DPSCs/ABCs at 1:1, 1:3, and 3:1 ratios displayed several crucial characteristics of odontoblast/ameloblast lineages, as indicated by accelerated mineralization, upregulated alkaline phosphatase activity, protein/gene expression for dentin sialophosphoprotein and ameloblastin. In vivo transplantation of reassociated DPSC and ABC pellets at different ratios was also carried out. Histological analyses demonstrated that only DPSC/ABC recombinants at 1:1 ratio generated typical molar crown-shaped structures, whereas recombinations at other ratios presented an atypical crown morphogenesis with unbalanced distribution of amelogenesis and dentinogenesis. Together, these findings revealed that the proportions of dental epithelial and mesenchymal cell populations can determine the odontogenic differentiation of DPSCs/ABCs in vitro as well as the bioengineered tooth morphogenesis in vivo.

Effects of FGF2 and TGFbeta1 on the differentiation of human dental pulp stem cells in vitro.

Two crucial growth factors, FGF2 and TGFbeta1, were investigated in this study to determine their inductive effects on the odontoblastic differentiation of human dental pulp stem cells (DPSCs) in vitro. DPSCs were isolated by immunomagnetic bead selection using the STRO-1 antibody, and then co-cultured respectively with FGF2, TGFbeta1 and FGF2+TGFbeta1. The results showed that FGF2 can exert a significant effect on the cell proliferation, while TGFbeta1 or FGF2+TGFbeta1 can initiate an odontoblast-like differentiation of DPSCs. Moreover, FGF2 can synergistically upregulate the effects of TGFbeta1 on the odontoblastic differentiation of DPSCs, as indicated by the increased alkaline phosphatase activity, the polarized cell appearance and secretary ultrastructural features, the formation of mineralized nodules and the gene/protein expression of dentin sialoprotein and dentin matrix protein-1. Together, FGF2 acted primarily on the cell proliferation, while TGFbeta1 and FGF2+TGFbeta1 mainly stimulated the odontoblastic differentiation of DPSCs. This study provides interesting progress in the odontoblastic differentiation of DPSCs induced by FGF2 and TGFbeta1.

[Experiment on inducing human dental pulp stem cells into neural-like cells].

OBJECTIVE: To explore the multi-differentiated capability of human dental pulp stem cells (hDPSCs) obtained by cell-clone culture approach and to determine the appropriate induced medium. METHODS: The cloned isolation and expansion of hDPSCs were preinduced for 24 h, and were subsequently replaced with neural-inductive medium containing certain concentration of dimethylsulfoxide (DMSO), butylated hydroxyanisode (BHA), forskolin, P-mercaptoethanol (p-ME) and hydrocortisone for 4 days. Then induced cells were analyzed by morphological observation, immnocytochemical staining for non-specific esterase (NSE) and glial fibrillary acidic protein (GFAP) expression, RT-PCR for GFAP mRNA. Meanwhile, the uninduced hDPSCs were used as negative control. RESULTS: The morphology of induced cells changed at the initial 12 h, and displayed a typical neuron-like cells at 24 h. There was a gradual increase in the number of these neuronal differentiated cells with continuous induction. Furthermore, immnocytochemical staining showed that the induced cell expressed NSE and GFAP, two marked enzymes of neuron cell. The GFAP mRNA was also detected in induced cells by RT-PCR assay. In contrast, the uninduced cells maintained its original appearance and had no expression on them. CONCLUSION: hDPSCs may possess potential of multiple-differentiation and may differentiate into neuron-like cells on certain inductive condition.