Research and development of microenvironment's influence on stem cells from the apical papilla - construction of novel research microdevices: tooth-on-a-chip.

Stem cells are crucial in tissue engineering, and their microenvironment greatly influences their behavior. Among the various dental stem cell types, stem cells from the apical papilla (SCAPs) have shown great potential for regenerating the pulp-dentin complex. Microenvironmental cues that affect SCAPs include physical and biochemical factors. To research optimal pulp-dentin complex regeneration, researchers have developed several models of controlled biomimetic microenvironments, ranging from in vivo animal models to in vitro models, including two-dimensional cultures and three-dimensional devices. Among these models, the most powerful tool is a microfluidic microdevice, a tooth-on-a-chip with high spatial resolution of microstructures and precise microenvironment control. In this review, we start with the SCAP microenvironment in the regeneration of pulp-dentin complexes and discuss research models and studies related to the biological process.

Massive and efficient encapsulation of single cells in monodisperse droplets and collagen-alginate microgels using a microfluidic device.

Single-cell manipulation is the key foundation of life exploration at individual cell resolution. Constructing easy-to-use, high-throughput, and biomimetic manipulative tools for efficient single-cell operation is quite necessary. In this study, a facile and efficient encapsulation of single cells relying on the massive and controllable production of droplets and collagen-alginate microgels using a microfluidic device is presented. High monodispersity and geometric homogeneity of both droplet and microgel generation were experimentally demonstrated based on the well-investigated microfluidic fabricating procedure. The reliability of the microfluidic platform for controllable, high-throughput, and improved single-cell encapsulation in monodisperse droplets and microgels was also confirmed. A single-cell encapsulation rate of up to 33.6% was achieved based on the established microfluidic operation. The introduction of stromal material in droplets/microgels for encapsulation provided single cells an in vivo simulated microenvironment. The single-cell operation achievement offers a methodological approach for developing simple and miniaturized devices to perform single-cell manipulation and analysis in a high-throughput and microenvironment-biomimetic manner. We believe that it holds great potential for applications in precision medicine, cell microengineering, drug discovery, and biosensing.

Substrate Stiffness of Bone Microenvironment Controls Functions of Pre-Osteoblasts and Fibroblasts In Vitro.

The formation of bone in a bone defect is accomplished by osteoblasts, while the over activation of fibroblasts promotes fibrosis. However, it is not clear how the extracellular matrix stiffness of the bone-regeneration microenvironment affects the function of osteoblasts and fibroblasts. This study aim to investigate the effect of bone-regeneration microenvironment stiffness on cell adhesion, cell proliferation, cell differentiation, synthesizing matrix ability and its potential mechanisms in mechanotransduction, in pre-osteoblasts and fibroblasts. Polyacrylamide substrates mimicking the matrix stiffness of different stages of the bone-healing process (15 kPa, mimic granulation tissue; 35 kPa, mimic osteoid; 150 kPa, mimic calcified bone matrix) were prepared. Mouse pre-osteoblasts MC3T3-E1 and mouse fibroblasts NIH3T3 were plated on three types of substrates, respectively. There were significant differences in the adhesion of pre-osteoblasts and fibroblasts on different polyacrylamide substrates. Runx2 expression increased with increasing substrate stiffness in pre-osteoblasts, while no statistical differences were found in the Acta2 expression in fibroblasts on three substrates. OPN expression in pre-osteoblasts, as well as Fn1 and Col1a1 expression in fibroblasts, decreased with increasing stiffness. The difference between the cell traction force generated by pre-osteoblasts and fibroblasts on substrates was also found. Our results indicated that substrate stiffness is a potent regulator of pre-osteoblasts and fibroblasts with the ability of promoting osteogenic differentiation of pre-osteoblasts, while having no effect on myofibroblast differentiation of fibroblasts.

Biomimetic Approaches in Cardiac Tissue Engineering: Replicating the Native Heart Microenvironment.

Cardiovascular diseases, including heart failure, pose significant challenges in medical practice, necessitating innovative approaches for cardiac repair and regeneration. Cardiac tissue engineering has emerged as a promising solution, aiming to develop functional and physiologically relevant cardiac tissue constructs. Replicating the native heart microenvironment, with its complex and dynamic milieu necessary for cardiac tissue growth and function, is crucial in tissue engineering. Biomimetic strategies that closely mimic the natural heart microenvironment have gained significant interest due to their potential to enhance synthetic cardiac tissue functionality and therapeutic applicability. Biomimetic approaches focus on mimicking biochemical cues, mechanical stimuli, coordinated electrical signaling, and cell-cell/cell-matrix interactions of cardiac tissue. By combining bioactive ligands, controlled delivery systems, appropriate biomaterial characteristics, electrical signals, and strategies to enhance cell interactions, biomimetic approaches provide a more physiologically relevant environment for tissue growth. The replication of the native cardiac microenvironment enables precise regulation of cellular responses, tissue remodeling, and the development of functional cardiac tissue constructs. Challenges and future directions include refining complex biochemical signaling networks, paracrine signaling, synchronized electrical networks, and cell-cell/cell-matrix interactions. Advancements in biomimetic approaches hold great promise for cardiovascular regenerative medicine, offering potential therapeutic strategies and revolutionizing cardiac disease modeling. These approaches contribute to the development of more effective treatments, personalized medicine, and improved patient outcomes. Ongoing research and innovation in biomimetic approaches have the potential to revolutionize regenerative medicine and cardiac disease modeling by replicating the native heart microenvironment, advancing functional cardiac tissue engineering, and improving patient outcomes.

Evolution of Biomimetic Approaches for Regenerative and Restorative Dentistry.

Biomimetics refers to human-made processes, substances, systems, or devices that imitate nature. The art and science of designing and building biomimetic apparatus are called biomimetics. This method can be widely used in dentistry to restore the structure and function of normal tooth structure. Traditional approaches to treating damaged and decayed teeth require more aggressive preparation to place a "strong," stiff restoration. The emphasis was made on the strength of the restoration as well as its function and mechanical properties, despite several disadvantages like tooth fracture, making future treatment more difficult and invasive. This review paper will seek to provide a clear explanation of its scope, different fields of biomimetic dentistry, and materials used in biomimetics that improve the strength of the tooth.

Nerve growth factor-basic fibroblast growth factor poly-lactide co-glycolid sustained-release microspheres and the small gap sleeve bridging technique to repair peripheral nerve injury.

We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique. Multiple growth factors play a synergistic role in promoting the repair of peripheral nerve injury; as a result, in this study, we added basic fibroblast growth factors to the microspheres to further promote nerve regeneration. First, in an in vitro biomimetic microenvironment, we developed and used a drug screening biomimetic microfluidic chip to screen the optimal combination of nerve growth factor/basic fibroblast growth factor to promote the regeneration of Schwann cells. We found that 22.56 ng/mL nerve growth factor combined with 4.29 ng/mL basic fibroblast growth factor exhibited optimal effects on the proliferation of primary rat Schwann cells. The successfully prepared nerve growth factor-basic fibroblast growth factor-poly-lactide-co-glycolid sustained-release microspheres were used to treat rat sciatic nerve transection injury using the small gap sleeve bridge technique. Compared with epithelium sutures and small gap sleeve bridging alone, the small gap sleeve bridging technique combined with drug-free sustained-release microspheres has a stronger effect on rat sciatic nerve transfection injury repair at the structural and functional level.

Microenvironmental reprogramming of human dermal papilla cells for hair follicle tissue engineering.

The restoration of hair-inductive potential in human dermal papilla cells (hDPCs) is a tremendous challenge for hair regeneration. Much of the research thus far has indicated that three-dimensional (3-D) culture shows improved efficacy in hair follicle (HF) neogenesis. However, mature HF cannot regenerate in an incomplete microenvironment. This study developed an optimized 3-D co-culture system to restore the hair-inductive characteristics of hDPCs by mimicking the in-vivo microenvironment. As a result, Matrigel-encapsulated hDPCs spontaneously formed into hDPC aggregates (hDPAs), which exhibited better activity, higher proliferation rates, and less apoptosis and hypoxia than the ultra-low attachment culture. Interestingly, the co-culture with the hair matrix cells and dermal sheath cup cells further enhanced the expression of hair regeneration-related genes of hDPAs compared to conditioned medium and improved mature HF induction. In addition, these hDPAs with higher hair inductivity could be produced on a large scale and easily separated for gene expression detection. Finally, the mRNA sequencing, PCR, and WB results showed that the co-culture biomimetic microenvironment stimulated the canonical Wnt signaling pathway and inhibited the BMP signaling pathway. Thus, this co-culture system will provide a reliable platform that allows high-throughput culture, testing, and harvesting of hDPAs for HF tissue engineering. STATEMENT OF SIGNIFICANCE: Extensive hair loss continues to be difficult to treat and causes significant patient morbidity. Hair follicle (HF) tissue engineering may seem to be a way out. However, the absence of the in-vivo microenvironment fails to regenerate mature hairs. This study systematically described a biomimetic co-culture approach to generate better quality human dermal papilla cell aggregates (hDPAs) with improved hair inductive properties, which can be further used for HF tissue engineering. The hDPC microenvironment was reprogrammed through the controllable formation of self-assembled organoids in Matrigel and the tri-culture with hair matrix cells and dermal sheath cup cells. This work indicates that the production of hDPAs could be readily scaled, in theory for large-scale assays, analyses, or therapeutic applications.

Cultivation of Encapsulated Primary Human B Lymphocytes: A First Step toward a Bioartificial Germinal Center.

Polyelectrolyte microcapsules based on sodium cellulose sulfate (SCS) and poly-diallyl-dimethyl-ammonium chloride (PDADMAC) have previously been proposed as a suitable ex vivo microenvironment for the cultivation and differentiation of primary human T lymphocytes. Here, the same system is investigated for the cultivation of human primary B cells derived from adult tonsillar tissue. Proliferation and differentiation into subtypes are followed and compared to suspension cultures of B cells from the same pool performed in parallel. Total cell expansion is somewhat lower in the capsules than in the suspension cultures. More importantly, however, the differentiation of the initially mainly memory B cells into various subtypes, in particular into plasma cell (PC), shows significant differences. Clearly, the microenvironment provided by the microcapsules is beneficial for an accelerated induction of a germinal center-like B cell phenotype and afterward supports the long-term survival of the PC cells. Then, varying the encapsulation conditions (i.e., presence of human serum and dedicated cytokines in the capsule core) provides a tool for finetuning the B cell response. Hence, this methodology is suggested to pave the way toward ex vivo development of human immune organoids.

[Islet biomimetic microenvironment constructed by chitosan oligosaccharide protects islets from hypoxia-induced damage by reducing intracellular reactive oxygen species].

Objective: Gelatin methacryloyl (GelMA)/hyaluronic acid methacryloyl (HAMA)/chitosan oligosaccharide (COS) hydrogel was used to construct islet biomimetic microenvironment, and to explore the improvement effect of GelMA/HAMA/COS on islet activity and function under hypoxia. Methods: Islets cultured on the tissue culture plate was set as the control group, on the GelMA/HAMA/COS hydrogel with COS concentrations of 0, 1, 5, 10, and 20 mg/mL respectively as the experimental groups. Scanning electron microscopy was used to observe the microscopic morphology, rheometer test to evaluate the gel-forming properties, contact angle to detect the hydrophilicity, and the biocompatibility was evaluated by the scaffold extract to L929 cells [using cell counting kit 8 (CCK-8) assay]. The islets were extracted from the pancreas of 8-week-old Sprague Dawley rats and the islet purity and function were identified by dithizone staining and glucose-stimulated insulin secretion (GSIS) assays, respectively. Islets were cultured under hypoxia (1%O 2) for 24, 48, and 72 hours, respectively. Calcein-acetyl methyl/propidium iodide (Calcein-AM/PI) staining was used to evaluate the effect of hypoxia on islet viability. Islets were cultured in GelMA/HAMA/COS hydrogels with different COS concentrations for 48 hours, and the reactive oxygen species kits were used to evaluate the antagonism of COS against islet reactive oxygen species production under normoxia (20%O 2) and hypoxia (1%O 2) conditions. Calcein-AM/PI staining was used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions. Islets were cultured in tissue culture plates (group A), GelMA/HAMA hydrogels (group B), and GelMA/HAMA/COS hydrogels (group C) for 48 hours, respectively. Immunofluorescence and GSIS assays were used to evaluate the effect of COS on islet activity under hypoxia (1%O 2) conditions, respectively. Results: GelMA/HAMA/COS hydrogel had a porous structure, the rheometer test showed that it had good gel-forming properties, and the contact angle test showed good hydrophilicity. CCK-8 assay showed that the hydrogel in each group had good biocompatibility. The isolated rat islets were almost round, with high islet purity and insulin secretion ability. Islets were treated with hypoxia for 24, 48, and 72 hours, Calcein-AM/PI staining showed that the number of dead cells gradually increased with time, which were significantly higher than those in the non-hypoxia-treated group ( P<0.001). Reactive oxygen staining showed that GelMA/HAMA/COS hydrogels with different COS concentrations could antagonize the production of reactive oxygen under normal oxygen and hypoxia conditions, and this ability was positively correlated with COS concentration. Calcein-AM/PI staining indicated that GelMA/HAMA/COS hydrogels with different COS concentrations could improve islet viability under hypoxia conditions, and cell viability was positively correlated with COS concentration. Immunofluorescence staining showed that GelMA/HAMA/COS hydrogel could promote the expression of islet function-related genes under hypoxia conditions. GSIS assay results showed that the insulin secretion of islets in hypoxia condition of group C was significantly higher than that of groups B and C ( P<0.05). Conclusion: GelMA/HAMA/COS hydrogel has good biocompatibility, promotes islet survival and function by inhibiting reactive oxygen species, and is an ideal carrier for building islet biomimetic microenvironment for islet culture and transplantation.

[Role of Stem Cells and Their Biomimetic Matrix Microenvironment in Regenerative Repair of Articular Cartilage: A Review].

It is difficult for the articular cartilage to self-heal any damage it may incur due to its lack of nerves and blood vessels. Development in stem cell technology provides new prospects for articular cartilage regeneration. Currently, stem cells from different sources and their diverse applications have demonstrated different degrees of therapeutic effect and potential in articular cartilage repair. However, stem cells are highly sensitive to their microenvironment. Therefore, more and more researchers are focusing their attention on regulating stem cells and thus accelerating cartilage regeneration through the biomimetic microenvironment constructed by biologically functional scaffolds. We reviewed in this paper the sources of the stem cells used for cartilage repair, the application method of these stem cells, as well as the therapeutic effect, mechanism and limitations in the application of stem cells synergizing with the biomimetic microenvironment in promoting articular cartilage repair and regeneration. We hoped to provide suggestions for practical clinical research in the design and improvement of biofunctional cartilage repair scaffolds that synergize with stem cells.

Engineered Biomimetic Neural Stem Cell Niche.

PURPOSE OF REVIEW: Neural stem cells (NSCs) have the potential to proliferate and differentiate into functional neurons, heightening their potential use for therapeutic applications. This review explores bioengineered systems which recapitulate NSC niche cell-cell and cell-matrix interactions. RECENT FINDINGS: Delivery of NSCs to the cytotoxic injured brain is limited by low cell survival rates post-transplantation and poor maintenance of native niche bioactive components. The use of biomaterial platforms can mimic in vivo the environment of the two germinal areas of the adult brain in which NSCs thrive. An environmental mimic that includes extracellular proteins and moieties, along with appropriate biomechanical cues has recently demonstrated promising results in enhancing neurogenesis, aiding the production of a bioengineered niche. SUMMARY: Biocomposition, biomechanics, and biostructure can be manipulated through engineered platforms to re-create the biofunctionality of an NSC niche. Upon transplantation and delivery with biomimetic scaffolds, NSCs show potential to promote functional recovery and rebuild neural circuitry post neurological trauma.

Scale-up of the ex vivo expansion of encapsulated primary human T lymphocytes.

A number of evolving medical therapies call for the controlled expansion of primary human T lymphocytes. After encapsulation in sodium cellulose sulfate-poly(diallyldimethyl) ammonium chloride polyelectrolyte capsules, T lymphocytes can be expanded without persisting activation. Here, the challenge of scaling up this process is addressed. Encapsulated T lymphocytes were cultured in spinner flasks as well as in several types of the bioreactor, including fixed and fluidized beds, a waved cell bag, and a standard stirred tank reactor (STR; 1-L scale). Two proprietary T lymphocyte culture media as well as a standard RPMI-based medium were used. As before, encapsulation coincided with the presence of only a low fraction of activated T lymphocytes (peripheral blood T cells) in the total population. Unexpectedly, growth rates were lower in well-mixed reactors than those in cultivations under static conditions, that is, in T-flasks. Switching the STR to low oxygen conditions (40% air saturation) improved the growth rates to the level of the static cultures and thus forms the potential basis for efficient scale-up of T lymphocyte expansion.

Creating a Biomimetic Microenvironment for the Ex Vivo Expansion of Primary Human T Lymphocytes.

The ex vivo expansion of primary human T cells is of considerable interest. Current protocols call for the addition of massive amounts of stimuli. This study presents as alternative the expansion of such cells in semipermeable sodium cellulose sulfate/poly(diallyldimethyl) ammonium chloride (SCS/PDADMAC) polyelectrolyte microcapsules, which supports at least six cell divisions and results in >40 × 106 cells mLcapsule-1 within less than 10 d. Inside the microcapsules, the T cells are suspended in a viscous SCS-solution. The low molecular weight cut off (<10 000 Da) of the surrounding polyelectrolyte membrane assures that typical signaling molecules produced by the cells are retained, while nutrients and metabolites can pass. Expensive additives, such as interleukin-2 (IL-2), can be coencapsulated. Expansion then no longer requires specialized T-cell media. Moreover, these results suggest that an SCS with a low degree of sulfation has biomimetic properties, representing an artificial extracellular matrix mimicking heparin sulfate.

Biomimetic 3D Tissue Models for Advanced High-Throughput Drug Screening.

Most current drug screening assays used to identify new drug candidates are 2D cell-based systems, even though such in vitro assays do not adequately re-create the in vivo complexity of 3D tissues. Inadequate representation of the human tissue environment during a preclinical test can result in inaccurate predictions of compound effects on overall tissue functionality. Screening for compound efficacy by focusing on a single pathway or protein target, coupled with difficulties in maintaining long-term 2D monolayers, can serve to exacerbate these issues when using such simplistic model systems for physiological drug screening applications. Numerous studies have shown that cell responses to drugs in 3D culture are improved from those in 2D, with respect to modeling in vivo tissue functionality, which highlights the advantages of using 3D-based models for preclinical drug screens. In this review, we discuss the development of microengineered 3D tissue models that accurately mimic the physiological properties of native tissue samples and highlight the advantages of using such 3D microtissue models over conventional cell-based assays for future drug screening applications. We also discuss biomimetic 3D environments, based on engineered tissues as potential preclinical models for the development of more predictive drug screening assays for specific disease models.