Cell-scaffold Constructs Research Articles

A Chitosan Scaffold Supports the Enhanced and Prolonged Differentiation of HiPSCs into Nucleus Pulposus-like Cells.

Intervertebral disc degeneration (IDD) is a progressive condition and stands as one of the primary causes of low back pain. Cell therapy that uses nucleus pulposus (NP)-like cells derived from human induced pluripotent stem cells (hiPSCs) holds great promise as a treatment for IDD. However, the conventional two-dimensional (2D) monolayer cultures oversimplify cell-cell interactions, leading to suboptimal differentiation efficiency and potential loss of phenotype. While three-dimensional (3D) culture systems like Matrigel improve hiPSC differentiation efficiency, they are limited by animal-derived materials for translation, poorly defined composition, short-term degradation, and high cost. In this study, we introduce a new 3D scaffold fabricated using medical-grade chitosan with a high degree of deacetylation. The scaffold features a highly interconnected porous structure, near-neutral surface charge, and exceptional degradation stability, benefiting iPSC adhesion and proliferation. This scaffold remarkably enhances the differentiation efficiency and allows uninterrupted differentiation for up to 25 days without subculturing. Notably, cells differentiated on the chitosan scaffold exhibited increased cell survival rates and upregulated gene expression associated with extracellular matrix secretion under a chemically defined condition mimicking the challenging microenvironment of intervertebral discs. These characteristics qualify the chitosan scaffold-cell construct for direct implantation, serving as both a structural support and a cellular source for enhanced stem cell therapy for IDD.

Bone Regeneration Using Mesenchymal Stromal Cells and Biocompatible Scaffolds: A Concise Review of the Current Clinical Trials.

Bone regenerative medicine is a clinical approach combining live osteoblast progenitors, such as mesenchymal stromal cells (MSCs), with a biocompatible scaffold that can integrate into host bone tissue and restore its structural integrity. Over the last few years, many tissue engineering strategies have been developed and thoroughly investigated; however, limited approaches have been translated to clinical application. Consequently, the development and clinical validation of regenerative approaches remain a centerpiece of investigational efforts towards the clinical translation of advanced bioengineered scaffolds. The aim of this review was to identify the latest clinical trials related to the use of scaffolds with or without MSCs to regenerate bone defects. A revision of the literature was performed in PubMed, Embase, and Clinicaltrials.gov from 2018 up to 2023. Nine clinical trials were analyzed according to the inclusion criteria: six presented in the literature and three reported in Clinicaltrials.gov. Data were extracted covering background trial information. Six of the clinical trials added cells to scaffolds, while three used scaffolds alone. The majority of scaffolds were composed of calcium phosphate ceramic alone, such as β-tricalcium phosphate (TCP) (two clinical trials), biphasic calcium phosphate bioceramic granules (three clinical trials), and anorganic bovine bone (two clinical trials), while bone marrow was the primary source of the MSCs (five clinical trials). The MSC expansion was performed in GMP facilities, using human platelet lysate (PL) as a supplement without osteogenic factors. Only one trial reported minor adverse events. Overall, these findings highlight the importance and efficacy of cell-scaffold constructs in regenerative medicine under different conditions. Despite the encouraging clinical results obtained, further studies are needed to assess their clinical efficacy in treating bone diseases to optimize their application.

IPSC-derived tenocytes seeded on microgrooved 3D printed scaffolds for Achilles tendon regeneration.

Tendons and ligaments have a poor innate healing capacity, yet account for 50% of musculoskeletal injuries in the United States. Full structure and function restoration postinjury remains an unmet clinical need. This study aimed to assess the application of novel three dimensional(3D) printed scaffolds and induced pluripotent stem cell-derived mesenchymal stem cells (iMSCs) overexpressing the transcription factor Scleraxis (SCX, iMSCSCX+ ) as a new strategy for tendon defect repair. The polycaprolactone (PCL) scaffolds were fabricated by extrusion through a patterned nozzle or conventional round nozzle. Scaffolds were seeded with iMSCSCX+ and outcomes were assessed in vitro via gene expression analysis and immunofluorescence. In vivo, rat Achilles tendon defects were repaired with iMSCSCX+ -seeded microgrooved scaffolds, microgrooved scaffolds only, or suture only and assessed via gait, gene expression, biomechanical testing, histology, and immunofluorescence.iMSCSCX+ -seeded on microgrooved scaffolds showed upregulation of tendon markers and increased organization and linearity of cells compared to non-patterned scaffolds in vitro. In vivo gait analysis showed improvement in the Scaffold + iMSCSCX+ -treated group compared to the controls. Tensile testing of the tendons demonstrated improved biomechanical properties of the Scaffold + iMSCSCX+ group compared with the controls. Histology and immunofluorescence demonstrated more regular tissue formation in the Scaffold + iMSCSCX+ group. This study demonstrates the potential of 3D-printed scaffolds with cell-instructive surface topography seeded with iMSCSCX+ as an approach to tendon defect repair. Further studies of cell-scaffold constructs can potentially revolutionize tendon reconstruction by advancing the application of 3D printing-based technologies toward patient-specific therapies that improve healing and functional outcomes at both the cellular and tissue level.

A comparative study on various cell sources for constructing tissue-engineered meniscus.

Injury to the meniscus is a common occurrence in the knee joint and its management remains a significant challenge in the clinic. Appropriate cell source is essential to cell-based tissue regeneration and cell therapy. Herein, three commonly used cell sources, namely, bone marrow mesenchymal stem cell (BMSC), adipose-derived stem cell (ADSC), and articular chondrocyte, were comparatively evaluated to determine their potential for engineered meniscus tissue in the absence of growth factor stimulus. Cells were seeded on electrospun nanofiber yarn scaffolds that share similar aligned fibrous configurations with native meniscus tissue for constructing meniscus tissue in vitro. Our results show that cells proliferated robustly along nanofiber yarns to form organized cell-scaffold constructs, which recapitulate the typical circumferential fiber bundles of native meniscus. Chondrocytes exhibited different proliferative characteristics and formed engineered tissues with distinct biochemical and biomechanical properties compared to BMSC and ADSC. Chondrocytes maintained good chondrogenesis gene expression profiles and produced significantly increased chondrogenic matrix and form mature cartilage-like tissue as revealed by typical cartilage lacunae. In contrast, stem cells underwent predominately fibroblastic differentiation and generated greater collagen, which contributes to improved tensile strengths of cell-scaffold constructs in comparison to the chondrocyte. ADSC showed greater proliferative activity and increased collagen production than BMSC. These findings indicate that chondrocytes are superior to stem cells for constructing chondrogenic tissues while the latter is feasible to form fibroblastic tissue. Combination of chondrocytes and stem cells might be a possible solution to construct fibrocartilage tissue and meniscus repair and regeneration.

Three-dimensional, label-free cell viability measurements in tissue engineering scaffolds using optical coherence tomography.

In the field of tissue engineering, 3D scaffolds and cells are often combined to yield constructs that are used as therapeutics to repair or restore tissue function in patients. Viable cells are often required to achieve the intended mechanism of action for the therapy, where the live cells may build new tissue or may release factors that induce tissue regeneration. Thus, there is a need to reliably measure cell viability in 3D scaffolds as a quality attribute of a tissue-engineered medical product. Here, we developed a noninvasive, label-free, 3D optical coherence tomography (OCT) method to rapidly (2.5min) image large sample volumes (1 mm3 ) to assess cell viability and distribution within scaffolds. OCT imaging was assessed using a model scaffold-cell system consisting of a polysaccharide-based hydrogel seeded with human Jurkat cells. Four test systems were used: hydrogel seeded with live cells, hydrogel seeded with heat-shocked or fixed dead cells and hydrogel without any cells. Time series OCT images demonstrated changes in the time-dependent speckle patterns due to refractive index (RI) variations within live cells that were not observed for pure hydrogel samples or hydrogels with dead cells. The changes in speckle patterns were used to generate live-cell contrast by image subtraction. In this way, objects with large changes in RI were binned as live cells. Using this approach, on average, OCT imaging measurements counted 326 ± 52 live cells per 0.288 mm3 for hydrogels that were seeded with 288 live cells (as determined by the acridine orange-propidium iodide cell counting method prior to seeding cells in gels). Considering the substantial uncertainties in fabricating the scaffold-cell constructs, such as the error from pipetting and counting cells, a 13% difference in the live-cell count is reasonable. Additionally, the 3D distribution of live cells was mapped within a hydrogel scaffold to assess the uniformity of their distribution across the volume. Our results demonstrate a real-time, noninvasive method to rapidly assess the spatial distribution of live cells within a 3D scaffold that could be useful for assessing tissue-engineered medical products.

Macrophages: From Simple Phagocyte to an Integrative Regulatory Cell for Inflammation and Tissue Regeneration-A Review of the Literature.

The understanding of macrophages and their pathophysiological role has dramatically changed within the last decades. Macrophages represent a very interesting cell type with regard to biomaterial-based tissue engineering and regeneration. In this context, macrophages play a crucial role in the biocompatibility and degradation of implanted biomaterials. Furthermore, a better understanding of the functionality of macrophages opens perspectives for potential guidance and modulation to turn inflammation into regeneration. Such knowledge may help to improve not only the biocompatibility of scaffold materials but also the integration, maturation, and preservation of scaffold-cell constructs or induce regeneration. Nowadays, macrophages are classified into two subpopulations, the classically activated macrophages (M1 macrophages) with pro-inflammatory properties and the alternatively activated macrophages (M2 macrophages) with anti-inflammatory properties. The present narrative review gives an overview of the different functions of macrophages and summarizes the recent state of knowledge regarding different types of macrophages and their functions, with special emphasis on tissue engineering and tissue regeneration.

Optimized Method of 3D Scaffold Seeding, Cell Cultivation, and Monitoring Cell Status for Bone Tissue Engineering.

The cultivation of cells in 3D systems is commonly regarded to be more physiological than in 2D as it comes much closer to the natural situation in tissues in many different aspects. However, 3D cell culture is much more complex. Cells within the pores of a printed 3D scaffold face a special situation concerning cell-material interaction and cell adhesion, cell proliferation, and supply of medium and oxygen into the core of the scaffolds. Biological assays (for cell proliferation, viability, and activity) have been validated primarily for 2D cell cultures and need to be adapted for 3D cultures. Likewise, in imaging, a number of points need to be taken into account in order to get a clear picture of the cells in 3D scaffolds, preferably with the method of multiphoton microscopy. Here, we describe a method for pretreatment and cell seeding of porous inorganic composite scaffolds (α-TCP/HA) for bone tissue engineering and for cultivation of the cell-scaffold constructs. The analytical methods described are the cell proliferation assay and the ALP activity assay. A step-by-step protocol is provided here that safely tackles typical difficulties that arise with this 3D cell-scaffold setting. In addition, MPM imaging of cells is described both with and without labeling. The combination of biochemical assays and imaging provides valuable insights into the possibilities of analysis with this 3D cell-scaffold system.

Development of osteon-like scaffold-cell construct by quadruple coaxial extrusion-based 3D bioprinting of nanocomposite hydrogel

Despite advances in bone tissue engineering, fabricating a scaffold which can be used as an implant for large bone defects remains challenge. One of the great importance in fabricating a biomimetic bone implant is considering the possibility of the integration of the structure and function of implants with hierarchical structure of bone. Herein, we propose a method to mimic the structural unit of compact bone, osteon, with spatial pattern of human umbilical vein endothelial cells (HUVECs) and human mesenchymal stem cells (hMSCs) in the adjacent layers that mimic Haversian canal and lamella, respectively. To this end, coaxial extrusion-based bioprinting technique via a customized quadruple-layer core-shell nozzle was employed. 3D implant scaffold-cell construct was fabricated by using polyethylene glycol as a hollowing agent in the first layer, gelatin methacryloyl (GelMA) and alginate blended hydrogel encapsulating HUVEC cells with vascular endothelial growth factor nanoparticles in the second layer (vasculogenic layer) to mimic vascular vessel, and GelMA and alginate blended hydrogel containing hMSCs cells in the outer osteogenic layer to imitate lamella. Two types of bone minerals, whitlockite and hydroxyapatite, were incorporated in osteogenic layer to induce osteoblastic differentiation and enhance mechanical properties (the young's modules of nanocomposite increased from 35 kPa to 80 kPa). In-vitro evaluations demonstrated high cell viability (94 % within 10 days) and proliferation. Furthermore, ALP enzyme activity increased considerably within 2 weeks and mineralized extra cellular matrix considerably produced within 3 weeks. Also, a significant increase in osteogenic markers was observed indicating the presence of differentiated osteoblast cells. Therefore, the work indicates the potential of single step 3D bioprinting process to fabricate biomimetic osteons to use as bone grafts for regeneration.

Evolution of functional tissue engineering for tendon and ligament repair.

This review paper is motivated by a Back-to-Basics presentation given by the author at the 2022 Orthopaedic Research Societymeeting in Tampa, Florida. I was tasked with providing a brief history of research leading up to the introduction of functional tissue engineering (FTE) for tendon and ligament repair. Beginning in the 1970s, this timeline focused on two common orthopedic soft tissue problems, anterior cruciate ligament ruptures in the knee and supraspinatus tendon injuries in the shoulder. Historic changes in the field over the next 5decades revealed a transformation from a focus more on mechanics (called "bioMECHANICS") on a larger (tissue) scale to a more recent focus on biology (called "mechanoBIOLOGY") on a smaller (cellular and molecular) scale. Early studies by surgeons and engineers revealed the importance of testing conditions for ligaments and tendons (e.g., high strain rates while avoiding subject disuse and immobility) and the need to measure in vivo forces in these tissues. But any true tissue engineering and regeneration in these early decades was limited more to the use of auto-, allo- and xenografts than actual generation of stimulated cell-scaffold constructs in culture. It was only after the discovery of tissue engineering in 1988 and the recognition of frequent rotator cuff injuries in the early 1990s, that biologists joined surgeons and engineers to discover mechanical and biological testing criteria for FTE. This review emphasizes the need for broader and more inclusive collaborations by surgeons, biologists and engineers in the short term with involvement of those in biomaterials, manufacturing, and regulation of new products in the longer term.

Integrated construction of silkworm cocoon-inspired 3D scaffold for improving cell manufacture and cryopreservation

Although cellular therapy holds enormous promise in treating intractable diseases, its application potential has been significantly hampered due to the scarcity of reliable and consistent cell sources. Therefore, a high-efficiency strategy that improves cell production and storage is desperately needed. Herein, we develop a versatile 3D bioinspired scaffold (Cryosilk) for improving scalable cell manufacture and cryopreservation. A bottom-up fabrication technique integrating electrospinning, in situ surface functionalization and freeze-shaping was explored to construct Cryosilk with biomimetic features and functions of silkworm cocoons. Cryosilk is composed of a core-shell heterostructure with silk fibroin/poly alanine fiber core and silk sericin shell, generating a 3D cocoon-mimicking fibrous structure. Importantly, Cryosilk possesses improved thermal conductivity and ice crystal resistance capability, thus enabling to cryopreserve biological samples with minimal cryodamage. Furthermore, Cryosilk not only promotes cell adhesion and growth, but achieves rapid and uniform rewarming process, which provides high cryopreservation efficacy for immune cells and stem cells. Particularly, Cryosilk can maintain cell viability and biofunctions of stem cell-scaffold constructs after freeze-thawing, which can be directly implanted to promote wound healing. Thus, Cryosilk offers unprecedented efficacy in cell manufacture and cryopreservation, which provides sufficient and high-quality precious cells and tissue engineered scaffolds for cellular therapy.

A Three-Dimensional Printed Polycaprolactone-Biphasic-Calcium-Phosphate Scaffold Combined with Adipose-Derived Stem Cells Cultured in Xenogeneic Serum-Free Media for the Treatment of Bone Defects.

The efficacy of a three-dimensional printed polycaprolactone–biphasic-calcium-phosphate scaffold (PCL–BCP TDP scaffold) seeded with adipose-derived stem cells (ADSCs), which were cultured in xenogeneic serum-free media (XSFM) to enhance bone formation, was assessed in vitro and in animal models. The ADSCs were isolated from the buccal fat tissue of six patients using enzymatic digestion and the plastic adherence method. The proliferation and osteogenic differentiation of the cells cultured in XSFM when seeded on the scaffolds were assessed and compared with those of cells cultured in a medium containing fetal bovine serum (FBS). The cell–scaffold constructs were cultured in XSFM and were implanted into calvarial defects in thirty-six Wistar rats to assess new bone regeneration. The proliferation and osteogenic differentiation of the cells in the XSFM medium were notably better than that of the cells in the FBS medium. However, the efficacy of the constructs in enhancing new bone formation in the calvarial defects of rats was not statistically different to that achieved using the scaffolds alone. In conclusion, the PCL–BCP TDP scaffolds were biocompatible and suitable for use as an osteoconductive framework. The XSFM medium could support the proliferation and differentiation of ADSCs in vitro. However, the cell–scaffold constructs had no benefit in the enhancement of new bone formation in animal models.

Alginate functionalized biomimetic 3D scaffold improves cell culture and cryopreservation for cellular therapy

The clinical translation of cellular therapy is hampered by the scarcity of reliable and consistent cell sources. In this study, we developed an exquisite scaffold featuring the hierarchical structure and biofunctions of silkworm cocoons (CryoSiCo), for boosting cell manufacture and cryopreservation. CryoSiCo was constructed by a creative bottom-up fabrication technique integrating electrospinning, in situ surface functionalization and freeze-shaping, generating a 3D cocoon-mimicking fibrous scaffold composed of graphene oxide-incorporated polylactic acid/gelatin inner fiber core and alginate outer fiber shell. CryoSiCo provided rapid and uniform rewarming for cryopreserved cells, and maximally maintained cell viability and proliferation capability, allowing for effective cryopreservation. Importantly, CryoSiCo could cryopreserve stem cell-scaffold constructs with high cell survival and functions, which can be directly implanted to restore tissue defects. Thus, CryoSiCo represents an appealing biomimetic strategy for storing precious cells and tissue engineered constructs, showing a broad application for fundamental research and applied medicine.

Scaffold Pore Curvature Influences ΜSC Fate through Differential Cellular Organization and YAP/TAZ Activity.

Tissue engineering aims to repair, restore, and/or replace tissues in the human body as an alternative to grafts and prostheses. Biomaterial scaffolds can be utilized to provide a three-dimensional microenvironment to facilitate tissue regeneration. Previously, we reported that scaffold pore size influences vascularization and extracellular matrix composition both in vivo and in vitro, to ultimately influence tissue phenotype for regenerating cranial suture and bone tissues, which have markedly different tissue properties despite similar multipotent stem cell populations. To rationally design biomaterials for specific cell and tissue fate specification, it is critical to understand the molecular processes governed by cell-biomaterial interactions, which guide cell fate specification. Building on our previous work, in this report we investigated the hypothesis that scaffold pore curvature, the direct consequence of pore size, modulates the differentiation trajectory of mesenchymal stem cells (MSCs) through alterations in the cytoskeleton. First, we demonstrated that sufficiently small pores facilitate cell clustering in subcutaneous explants cultured in vivo, which we previously reported to demonstrate stem tissue phenotype both in vivo and in vitro. Based on this observation, we cultured cell-scaffold constructs in vitro to assess early time point interactions between cells and the matrix as a function of pore size. We demonstrate that principle curvature directly influences nuclear aspect and cell aggregation in vitro. Scaffold pores with a sufficiently low degree of principle curvature enables cell differentiation; pharmacologic inhibition of actin cytoskeleton polymerization in these scaffolds decreased differentiation, indicating a critical role of the cytoskeleton in transducing cues from the scaffold pore microenvironment to the cell nucleus. We fabricated a macropore model, which allows for three-dimensional confocal imaging and demonstrates that a higher principle curvature facilitates cell aggregation and the formation of a potentially protective niche within scaffold macropores which prevents MSC differentiation and retains their stemness. Sufficiently high principle curvature upregulates yes-associated protein (YAP) phosphorylation while decreased principle curvature downregulates YAP phosphorylation and increases YAP nuclear translocation with subsequent transcriptional activation towards an osteogenic differentiation fate. Finally, we demonstrate that the inhibition of the YAP/TAZ pathway causes a defect in differentiation, while YAP/TAZ activation causes premature differentiation in a curvature-dependent way when modulated by verteporfin (VP) and 1-oleyl-lysophosphatidic acid (LPA), respectively, confirming the critical role of biomaterials-mediated YAP/TAZ signaling in cell differentiation and fate specification. Our data support that the principle curvature of scaffold macropores is a critical design criterion which guides the differentiation trajectory of mesenchymal stem cells’ scaffolds. Biomaterial-mediated regulation of YAP/TAZ may significantly contribute to influencing the regenerative outcomes of biomaterials-based tissue engineering strategies through their specific pore design.

Hydrostatic pressure stimulates the osteogenesis and angiogenesis of MSCs/HUVECs co-culture on porous PLGA scaffolds

In native bone tissue regeneration, blood vessels, providing oxygen and nutrition for tissues, can promote the regeneration of bone and accelerate the repair of a defected area. In this study, Poly(D, L-lactic-co-glycolic acid) (PLGA) inverse opal scaffolds with high pore interconnectivity were fabricated and further modified with vascular endothelial growth factor (VEGF). The rat bone marrow derived mesenchymal stem cells (rMSCs) and human umbilical vein endothelial cells (HUVECs) were co-cultured onto the scaffolds to enhance vascularization for bone tissue regeneration. Cell attachment, viability, proliferation, and morphology were detected by cell counting kit-8 (CCK-8) assay, live and dead staining and scanning electron microscopy (SEM). Hydrostatic pressure with 0–279 KPa and 1 Hz one hour per day for 7 days was applied to tissue engineered bone constructs to investigate whether the loading stimulation can promote osteogenesis and angiogenesis mutually evaluated in parallel by multiple in vitro assays and in an in vivo chicken chorioallantoic membrane (CAM) model. The results indicated that the immobilization of VEGF can improve biocompatibility of PLGA scaffolds and promote cell attachment and proliferation. The cell-scaffold constructs showed higher CD31 expression because of the angiogenic differentiation of rMSCs in hydrostatic loading culture condition in vitro. The in vivo CAM model experiment demonstrated that hydrostatic loading stimulated angiogenic differentiation of rMSCs can accelerate tubulogenesis. Furthermore, the new capillaries formed in cell-scaffold constructs were conducive to calcium deposition in vivo.

Transplantation of decellularised human amniotic membranes seeded with mesenchymal stem cell-educated macrophages into animal models.

The reconstruction of chronic skin wounds remains a public health challenge in dermatology. Precisely controlling and monitoring the wound-healing process should result in enhanced outcomes for the patient. Cell-based therapies have shown great potential in medicine due to their immunomodulatory and healing properties. Herein, we produced activated macrophages by treating circulating monocytes with mesenchymal stem cell (MSC) supernatant. We also demonstrated the critical role of activated macrophages transplantation using amniotic membranes in accelerating wound healing in an animal wound model. The activated macrophages not only exhibited immunomodulatory cytokines like transforming growth factorβ (TGFβ) and interleukin 10 (and IL10) secretion but also showed attachment and proliferation ability on the amniotic membrane scaffold. Moreover, MSCs supernatant-treated cells also displayed significant ARG1, CD206, and IL 10 genes expression. Inspired by the in vitro results, we examined the in vivo therapeutic efficacy of the activated macrophage transplantation using an acellular amniotic membrane carrier in a full-thickness cutaneous wound model. The wound healing rate was significant in the group treated with macrophages generated via mesenchymal cell therapy seeded human amniotic membrane. There was less scarring in the wound sites after placing cell-scaffold constructs in the wound sites in the animal models. Overall, macrophages stimulated with mesenchymal cells' supernatant exhibited improved healing processes in incisional wounds by decreasing the inflammatory phase, increasing angiogenesis, and reducing scar tissue development.

Generation of hybrid tissue engineered construct through embedding autologous chondrocyte loaded platelet rich plasma/alginate based hydrogel in porous scaffold for cartilage regeneration

Over the past decades, various attempts have been made to develop suitable tissue-engineered constructs to repair or regenerate the damaged or diseased articular cartilage. In the present study, we embedded Platelet rich plasma (PRP)/Sodium Alginate (SA) based hydrogel in porous 3D scaffold of chitosan (CH)/chondroitin sulfate (CS)/silk fibroin (SF) to develop hybrid scaffold for cartilage tissue construct generation with abilities to support shape recovery potential, facilitate uniform cells distribution and mimic gel like cartilage tissue extracellular matrix.The developed hybrid matrix shows suitable pore size (55–261 μm), porosity (77 ± 4.3%) and compressive strength (0.13 ± 0.04 MPa) for cartilage tissue construct generation and its applications. The developed SA/PRP-based cartilage construct exhibits higher metabolic activity, glycosaminoglycan deposition, expression of collagen type II, and aggrecan in comparison to SA based cell-scaffold construct. In-vivo animal study was also performed to investigate the biocompatibility and cartilage tissue regeneration potential of the developed construct. The obtained gross analysis of knee sample, micro-computed tomography, and histological analysis suggest that implanted tissue construct possess the superior potential to regenerate hyaline cartilage defect of thickness around 1.10 ± 0.36 mm and integrate with surrounding tissue at the defect site. Thus, the proposed strategy for the development of cartilage tissue constructs might be beneficial for the repair of full-thickness knee articular cartilage defects.

Mesenchymal Stem Cell Seeding on 3D Scaffolds.

Evaluation of mesenchymal stem cell seeding efficiency in three-dimensional (3D) scaffolds is a critical step for constructing a potent and useful tissue engineering product for regenerative medicine. To determine the quantity of cells seeded on a scaffold, their condition and viability, and/or to confirm cell adhesion to the scaffold surface, a number of cellular assays are used. The assays are most often based on a direct or indirect colorimetric-, fluorimetric-, bioluminescent-, or isotope-based measurement of changes reflecting the activity of cellular processes. This chapter presents a selection of assays measuring the efficiency of cell seeding on scaffolds, that is, the MTS (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium)) assay, the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, the ATP (adenosine triphosphate), DAPI (4',6-diamidino-2-phenylindole) assay, the Alamar Blue (7-hydroxy-10-oxidophenoxazin-10-ium-3-one, resazurin) assay and the Pico Green dsDNA (N'-[3-(dimethylamino)propyl]-N,N-dimethyl-N'-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]propane-1,3-diamine) assay. These assays monitor the number of viable cells, sometimes in conjunction with specifying cell membrane integrity, determine enzymatic activity associated with cell metabolism, measure cell proliferation rate, and assess the total protein or DNA content in the cell-scaffold construct. The choice of the appropriate methods and the details for testing 3D cultures are of utmost importance to properly evaluate tissue engineering products. Still, developing standards for assessment of cell-scaffold constructs remains a challenge in tissue engineering.

Converging 3D Printing and Electrospinning: Effect of Poly(l-lactide)/Gelatin Based Short Nanofibers Aerogels on Tracheal Regeneration.

Recently, various tissue engineering based strategies have been pursued for the regeneration of tracheal tissues. However, previously developed tracheal scaffolds do not accurately mimic the microstructure and mechanical behavior of the native trachea, which restrict their clinical translation. Here, tracheal scaffolds are fabricated by using 3D printing and short nanofibers (SF) dispersion of poly(l-lactide)/gelatin (0.5-1.5 wt%) to afford tracheal constructs. The results display that the scaffolds containing 1.0wt % of SF exhibit low density, good water absorption capacity, reasonable degradation rate, and stable mechanical properties, which were comparable to the native trachea. Moreover, the designed scaffolds possess good biocompatibility and promote the growth and infiltration of chondrocytes in vitro. The biocompatibility of tracheal scaffolds is further assessed after subcutaneous implantation in mice for up to 4 and 8 weeks. Histological assessment of tracheal constructs explanted at week 4 shows that scaffolds can maintain their structural integrity and support the formation of neo-vessels. Furthermore, cell-scaffold constructs gradually form cartilage-like tissues, which mature with time. Collectively, these engineered tracheal scaffolds not only possess appropriate mechanical properties to afford a stabilized structure but also a biomimetic extracellular matrix-like structure to accomplish tissue regeneration, which may have broad implications for tracheal regeneration.

Evaluation of the influence of platelet-rich plasma (PRP), platelet lysate (PL) and mechanical loading on chondrogenesis in vitro

The aim of this work is to investigate the capability of PRP as an adjuvant therapy to autologous chondrocyte implantation (ACI) in combination with multi-axial load with respect to cartilage regeneration. Articular cartilage shows poor repair capacity and therapies for cartilage defects are still lacking. Well-established operative treatments include ACI, and growing evidence shows the beneficial effects of PRP. Platelets contain numerous growth factors, among them transforming growth factor beta (TGF-β). Dynamic mechanical loading is known to be essential for tissue formation, improving extracellular matrix (ECM) production. For our ACI model monolayer expanded human chondrocytes were seeded into polyurethane scaffolds and embedded in fibrin (hChondro), in PRP-Gel (PRP), or in fibrin with platelet lysate (PL), which was added to the media once a week with a concentration of 50 vol%. The groups were either exposed to static conditions or multi-axial forces in a ball-joint bioreactor for 1 h per day over 2 weeks, mimicking ACI under physiological load. The culture medium was collected and analyzed for glycosaminoglycan (GAG), nitrite and transforming growth factor beta 1 (TGF-β1) content. The cell-scaffold constructs were collected for DNA and GAG quantification; the expression of chondrogenic genes, TGF-β and related receptors, as well as inflammatory genes, were analyzed using qPCR. Loading conditions showed superior chondrogenic differentiation (upregulation of COL2A1, ACAN, COMP and PRG4 expression) than static conditions. PRP and PL groups combined with mechanical loading showed upregulation of COL2A1, ACAN and COMP. The highest amount of total TGF-β1 was quantified in the PL group. Latent TGF-β1 was activated in all loaded groups, while the highest amount was found in the PL group. Load increased TGFBR1/TGFBR2 mRNA ratio, with further increases in response to supplements. In general, loading increased nitrite release into the media. However, over time, the media nitrite content was lower in the PL group compared to the control group. Based on these experiments, we conclude that chondrogenic differentiation is strongest when simulated ACI is performed in combination with dynamic mechanical loading and PRP-gel or PL supplementation. An inflammatory reaction was reduced by PRP and PL, which could be one of the major therapeutic effects. Loading presumably can enhance the action of TGF-β1, which was predominantly activated in loaded PL groups. The combination of load and PRP represents an effective and promising synergy concerning chondrocyte-based cartilage repair.

Clinical Applications of Cell-Scaffold Constructs for Bone Regeneration Therapy.

Bone tissue engineering (BTE) is a process of combining live osteoblast progenitors with a biocompatible scaffold to produce a biological substitute that can integrate into host bone tissue and recover its function. Mesenchymal stem cells (MSCs) are the most researched post-natal stem cells because they have self-renewal properties and a multi-differentiation capacity that can give rise to various cell lineages, including osteoblasts. BTE technology utilizes a combination of MSCs and biodegradable scaffold material, which provides a suitable environment for functional bone recovery and has been developed as a therapeutic approach to bone regeneration. Although prior clinical trials of BTE approaches have shown promising results, the regeneration of large bone defects is still an unmet medical need in patients that have suffered a significant loss of bone function. In this present review, we discuss the osteogenic potential of MSCs in bone tissue engineering and propose the use of immature osteoblasts, which can differentiate into osteoblasts upon transplantation, as an alternative cell source for regeneration in large bone defects.