Posttranscriptional Modification to Modulate Progenitor Differentiation on Heterotypic Spheroids.

Cell aggregates are widely used to study heterotypic cellular interactions during the development of vascularization in vitro. In this study, we examined heterotypic cellular spheroids made of adipose-derived stem cells and CD34+/CD31- endothelial progenitor cells induced by the transfection of miR-148b mimic for de novo induction of osteogenic differentiation and miR-210 mimic for de novo induction of endotheliogenesis, respectively. The effect of the microRNA (miRs) mimic treatment group and induction time on codifferentiation was assessed in spheroids formed of transfected cells over the course of a 4-week culture. Based on gene and protein markers of osteogenic and endotheliogenic differentiation, as well as mineralization assays, our results showed that miRs directed cell differentiation and that progenitor maturity influenced the development of heterotypic cellular regions in aggregates. Overall, the success of coculture to create a prevascularized bone model is dependent on a number of factors, particularly the induction time of differentiation before combining the multiple cell types in aggregates. The approach that has been proposed could be valuable in creating vascularized bone tissue by employing spheroids as the building blocks of more complex issues through the use of cutting-edge methods such as 3D bioprinting.

Ushering in the era of regenerative surgery.

Tissue loss, irrespective of etiology, often requires extensive reconstruction. In many instances, the need exceeds what current treatments and technologies modern medicine can offer. Tissue engineering has made immense strides within the past few decades due to advances in biologics, biomaterials, and manufacturing. The convergence of these three domains has created limitless potential for future surgical care. Unfortunately, there still exists a disconnect on how to best implant these 'replacement parts' and care for the patient. It is therefore vital to develop paradigms for the integration of advanced surgical and tissue engineering technologies. This paper explores the convergence between tissue engineering and reconstructive surgery. We will describe the clinical problem of tissue loss, discuss currently available solutions, address limitations, and propose processes for integrating surgery and tissue engineering, thereby ushering in the era of regenerative surgery.

Vascular persistence following precision micropuncture.

OBJECTIVE: The success of engineered tissues continues to be limited by time to vascularization and perfusion. Recently, we described a simple microsurgical approach, termed micropuncture (MP), which could be used to rapidly vascularize an adjacently placed scaffold from the recipient macrovasculature. Here we studied the long-term persistence of the MP-induced microvasculature. METHODS: Segmental 60 µm diameter MPs were created in the recipient rat femoral artery and vein followed by coverage with a simple Type 1 collagen scaffold. The recipient vasculature and scaffold were then wrapped en bloc with a silicone sheet to isolate intrinsic vascularization. Scaffolds were harvested at 28 days post-implantation for detailed analysis, including using a novel artificial intelligence (AI) approach. RESULTS: MP scaffolds demonstrated a sustained increase of vascular density compared to internal non-MP control scaffolds (p < 0.05) secondary to increases in both vessel diameters (p < 0.05) and branch counts (p < 0.05). MP scaffolds also demonstrated statistically significant increases in red blood cell (RBC) perfused lumens. CONCLUSIONS: This study further highlights that the intrinsic MP-induced vasculature continues to persist long-term. Its combination of rapid and stable angiogenesis represents a novel surgical platform for engineered scaffold and graft perfusion.

Accelerating Patterned Vascularization Using Granular Hydrogel Scaffolds and Surgical Micropuncture.

Bulk hydrogel scaffolds are common in reconstructive surgery. They allow for the staged repair of soft tissue loss by providing a base for revascularization. Unfortunately, they are limited by both slow and random vascularization, which may manifest as treatment failure or suboptimal repair. Rapidly inducing patterned vascularization within biomaterials has profound translational implications for current clinical treatment paradigms and the scaleup of regenerative engineering platforms. To address this long-standing challenge, a novel microsurgical approach and granular hydrogel scaffold (GHS) technology are co-developed to hasten and pattern microvascular network formation. In surgical micropuncture (MP), targeted recipient blood vessels are perforated using a microneedle to accelerate cell extravasation and angiogenic outgrowth. By combining MP with an adjacent GHS with precisely tailored void space architecture, microvascular pattern formation as assessed by density, diameter, length, and intercapillary distance is rapidly guided. This work opens new translational opportunities for microvascular engineering, advancing reconstructive surgery, and regenerative medicine.

Intraoperative bioprinting of human adipose-derived stem cells and extra-cellular matrix induces hair follicle-like downgrowths and adipose tissue formation during full-thickness craniomaxillofacial skin reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2 % or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo. Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

Intraoperative Bioprinting of Human Adipose-derived Stem cells and Extra-cellular Matrix Induces Hair Follicle-Like Downgrowths and Adipose Tissue Formation during Full-thickness Craniomaxillofacial Skin Reconstruction.

Craniomaxillofacial (CMF) reconstruction is a challenging clinical dilemma. It often necessitates skin replacement in the form of autologous graft or flap surgery, which differ from one another based on hypodermal/dermal content. Unfortunately, both approaches are plagued by scarring, poor cosmesis, inadequate restoration of native anatomy and hair, alopecia, donor site morbidity, and potential for failure. Therefore, new reconstructive approaches are warranted, and tissue engineered skin represents an exciting alternative. In this study, we demonstrated the reconstruction of CMF full-thickness skin defects using intraoperative bioprinting (IOB), which enabled the repair of defects via direct bioprinting of multiple layers of skin on immunodeficient rats in a surgical setting. Using a newly formulated patient-sourced allogenic bioink consisting of both human adipose-derived extracellular matrix (adECM) and stem cells (ADSCs), skin loss was reconstructed by precise deposition of the hypodermal and dermal components under three different sets of animal studies. adECM, even at a very low concentration such as 2% or less, has shown to be bioprintable via droplet-based bioprinting and exhibited de novo adipogenic capabilities both in vitro and in vivo . Our findings demonstrate that the combinatorial delivery of adECM and ADSCs facilitated the reconstruction of three full-thickness skin defects, accomplishing near-complete wound closure within two weeks. More importantly, both hypodermal adipogenesis and downgrowth of hair follicle-like structures were achieved in this two-week time frame. Our approach illustrates the translational potential of using human-derived materials and IOB technologies for full-thickness skin loss.

Mesenchymal Stem Cell-Derived Extracellular Vesicles for Therapeutic Use and in Bioengineering Applications.

Extracellular vesicles (EVs) are small lipid bilayer-delimited particles that are naturally released from cells into body fluids, and therefore can travel and convey regulatory functions in the distal parts of the body. EVs can transmit paracrine signaling by carrying over cytokines, chemokines, growth factors, interleukins (ILs), transcription factors, and nucleic acids such as DNA, mRNAs, microRNAs, piRNAs, lncRNAs, sn/snoRNAs, mtRNAs and circRNAs; these EVs travel to predecided destinations to perform their functions. While mesenchymal stem cells (MSCs) have been shown to improve healing and facilitate treatments of various diseases, the allogenic use of these cells is often accompanied by serious adverse effects after transplantation. MSC-produced EVs are less immunogenic and can serve as an alternative to cellular therapies by transmitting signaling or delivering biomaterials to diseased areas of the body. This review article is focused on understanding the properties of EVs derived from different types of MSCs and MSC-EV-based therapeutic options. The potential of modern technologies such as 3D bioprinting to advance EV-based therapies is also discussed.

Smooth Versus Textured Tissue Expander Breast Reconstruction: Complications and Efficacy.

INTRODUCTION: Ongoing recognition of breast implant-associated anaplastic large cell lymphoma (BIA-ALCL) and its link with textured devices has brought a paradigm shift in prosthetic-based breast reconstruction. Many institutions no longer offer textured expansion devices for staged reconstruction. However, there is a paucity of data regarding the efficacy of smooth tissue expanders (TE). We hypothesized that the time to final reconstruction and complication profile between smooth and textured TEs would be similar in breast reconstruction patients. METHODS: A retrospective chart review was performed of all patients who underwent TE breast reconstruction during a 6-year period at the Penn State Hershey Medical Center. Rates of complications treated nonoperatively and those requiring reoperation were assessed. Mechanical complications, including expander malposition and rupture, were evaluated. Time to final breast reconstruction was quantified. Mixed-effects logistic regression and linear regression models, as appropriate, were used to compare textured to smooth TEs. Patient characteristics and anatomic plane placement were adjusted for in all analyses of outcomes. RESULTS: Data were collected on 389 patients, encompassing 140 smooth and 604 textured TEs. Textured devices had an increased incidence of complications treated nonsurgically (16.7% vs 10.7%; P = 0.14). However, smooth TEs had an increased incidence of reoperation (12.1% vs 7.6%; P = 0.06). Most noteworthy was that although smooth TEs had a 40-fold increase in malposition (13.6% vs 0.3%; P < 0.001), no reoperation for this complication was warranted. Further, the time to final reconstruction was comparable between the 2 devices (textured expanders: 221 days and smooth expanders: 234 days; P = 0.15). CONCLUSIONS: Staged, implant-based reconstruction is the most common surgical approach to recreate the breast mound following mastectomy. Textured TEs were the cornerstone to this approach. Unfortunately, the association between textured devices and BIA-ALCL now mandates an alternative. We postulated that smooth expanders would compare favorably for breast reconstruction. Although our study suggests that smooth TEs suffer more malposition, this has a negligible impact on the reconstructive timeline. Thus, smooth TEs may prove beneficial when considering the risk of BIA-ALCL associated with textured devices.

Hybrid adipose graft materials synthesized from chemically modified adipose extracellular matrix.

Decellularized extracellular matrix (ECM) from tissues is a promising biomaterial that can provide a complex 3D microenvironment capable of modulating cell response and tissue regeneration. In this study, we have integrated the decellularized thiolated adipose-derived ECM, at different concentrations, with polyethylene glycol (PEG) using Michael addition between thiol and acrylate moieties. The potential for this material to support adipogenic differentiation of human adipose-derived stem cells was evaluated by encapsulating cells in hydrogels with increasing concentrations of chemically modified ECM (mECM). Our results demonstrated a positive correlation between the ECM content in the hydrogels and cell proliferation, adipogenic marker expression, and lipid formation and accumulation. Furthermore, we have shown host cell infiltration and enhanced adipogenesis in vivo after implantation. These findings support the graft as a potential alternative for adipose tissue regeneration.

Regenerative Engineering: Current Applications and Future Perspectives.

Many pathologies, congenital defects, and traumatic injuries are untreatable by conventional pharmacologic or surgical interventions. Regenerative engineering represents an ever-growing interdisciplinary field aimed at creating biological replacements for injured tissues and dysfunctional organs. The need for bioengineered replacement parts is ubiquitous among all surgical disciplines. However, to date, clinical translation has been limited to thin, small, and/or acellular structures. Development of thicker tissues continues to be limited by vascularization and other impediments. Nevertheless, currently available materials, methods, and technologies serve as robust platforms for more complex tissue fabrication in the future. This review article highlights the current methodologies, clinical achievements, tenacious barriers, and future perspectives of regenerative engineering.

Navigating the Genomic Landscape of Human Adipose Stem Cell-Derived ß-Cells.

Diabetes is a pandemic manifested through glucose dysregulation mediated by inadequate insulin secretion by beta cells. A beta cell replacement strategy would transform the treatment paradigm from pharmacologic glucose modulation to a genuine cure. Stem cells have emerged as a potential source for beta cell (ß-cell) engineering. The detailed generation of functional ß-cells from both embryonic and induced pluripotent stem cells has recently been described. Adult stem cells, including adipose derived, may also offer a therapeutic approach, but remain ill defined. In our study, we performed an in-depth assessment of insulin-producing beta cells generated from human adipose, irrespective of donor patient age, gender, and health status. Cellular transformation was confirmed using flow cytometry and single-cell imaging. Insulin secretion was observed with glucose stimulation and abrogated following palmitate exposure, a common free fatty acid implicated in human beta cell dysfunction. We used next-generation sequencing to explore gene expression changes before and after differentiation of patient-matched samples, which revealed more than 5,000 genes enriched. Adipose-derived beta cells displayed comparable gene expression to native ß-cells. Pathway analysis demonstrated relevance to stem cell differentiation and pancreatic developmental processes, which are vital to cellular function, structural development, and regulation. We conclude that the functions associated with adipose derived beta cells are mediated through relevant changes in the transcriptome, which resemble those seen in native ß-cell morphogenesis and maturation. Therefore, they may represent a viable option for the clinical translation of stem cell-based therapies in diabetes.

Intra-Operative Bioprinting of Hard, Soft, and Hard/Soft Composite Tissues for Craniomaxillofacial Reconstruction.

Reconstruction of complex craniomaxillofacial (CMF) defects is challenging due to the highly organized layering of multiple tissue types. Such compartmentalization necessitates the precise and effective use of cells and other biologics to recapitulate the native tissue anatomy. In this study, intra-operative bioprinting (IOB) of different CMF tissues, including bone, skin, and composite (hard/soft) tissues, is demonstrated directly on rats in a surgical setting. A novel extrudable osteogenic hard tissue ink is introduced, which induced substantial bone regeneration, with ≈80% bone coverage area of calvarial defects in 6 weeks. Using droplet-based bioprinting, the soft tissue ink accelerated the reconstruction of full-thickness skin defects and facilitated up to 60% wound closure in 6 days. Most importantly, the use of a hybrid IOB approach is unveiled to reconstitute hard/soft composite tissues in a stratified arrangement with controlled spatial bioink deposition conforming the shape of a new composite defect model, which resulted in ≈80% skin wound closure in 10 days and 50% bone coverage area at Week 6. The presented approach will be absolutely unique in the clinical realm of CMF defects and will have a significant impact on translating bioprinting technologies into the clinic in the future.

Computer-Aided Design and Manufacture of Intraoral Splints: A Potential Role in Cleft Care.

BACKGROUND: Nasoalveolar molding is a nonsurgical modality for the treatment of cleft lip and palate that uses an intraoral splint to align the palatal shelves. Repeated impressions are needed for splint modification, each carrying risk of airway obstruction. Computer-aided design and manufacturing (CAD/CAM) has the ability to simplify the process. As a precursor to CAD/CAM splint fabrication, a proof-of-concept study was conducted to compare three-dimensional splints printed from alginate impressions versus digital scans. We hypothesized that intraoral digital scanning would compare favorably to alginate impressions for palate registration and subsequent splint manufacture, with decreased production times. METHODS: Alginate and digital impressions were taken from 25 healthy teenage volunteers. Digital impressions were performed with a commercially available intraoral scanner. Plaster casts made from alginate impressions were converted to Standard Triangle Language files. Patient-specific matched scans were evaluated for total surface area with the concordance correlation coefficient. Acrylic palatal splints were three-dimensionally printed from inverse digital molds. Subjective appliance fit was assessed using a five-point scale. RESULTS: A total of 23 participants were included. Most subjects preferred digital impression acquisition. Impression methods showed moderate agreement (concordance correlation coefficient 0.93). Subjects rated splints from digital impressions as having a more precise fit (4.4 versus 3.9). The digital approach decreased impression phase time by over 10-fold and overall production time by 28%. CONCLUSIONS: CAD/CAM has evolved extensively over the past two decades and is now commonplace in medicine. However, its utility in cleft patients has not been fully realized. This pilot study demonstrated that CAD/CAM technologies may prove useful in patients requiring intraoral splints.

Induction of scaffold angiogenesis by recipient vasculature precision micropuncture.

The success of engineered tissues continues to be limited by time to vascularization and perfusion. Here, we studied the effects of precision injury to a recipient macrovasculature in promoting neovessel formation in an adjacently placed scaffold. Segmental 60 µm diameter micropunctures (MP) were created in the recipient rat femoral artery and vein followed by coverage with a simple collagen scaffold. Scaffolds were harvested at 24, 48, 72, and 96 h post-implantation for detailed analysis. Those placed on top of an MP segment showed an earlier and more robust cellular infiltration, including both endothelial cells (CD31) and macrophages (F4/80), compared to internal non-micropunctured control limbs (p < 0.05). At the 96-hour timepoint, MP scaffolds demonstrated an increase in physiologic perfusion (p < 0.003) and a 2.5-fold increase in capillary network formation (p < 0.001). These were attributed to an overall upsurge in small vessel quantity. Furthermore, MP positioned scaffolds demonstrated significant increases in many modulators of angiogenesis, including VEGFR2 and Tie-2 despite a decrease in HIF-1α at all timepoints. This study highlights a novel microsurgical approach that can be used to rapidly vascularize or inosculate contiguously placed scaffolds and grafts. Thereby, offering an easily translatable route towards the creation of thicker and more clinically relevant engineered tissues.

Differentiation of Adipose Tissue-Derived CD34+/CD31- Cells into Endothelial Cells In Vitro.

In this study, CD34+/CD31- progenitor cells were isolated from the stromal vascular fraction (SVF) of adipose tissue using magnetic activated cell sorting. The endothelial differentiation capability of these cells in vitro was evaluated by culturing them in vascular endothelial growth factor (VEGF) induced medium for 14 days. Viability, proliferation, differentiation and tube formation of these cells were evaluated. Cell viability study revealed that both undifferentiated and endothelial differentiated cells remained healthy for 14 days. However, the proliferation rate was higher in undifferentiated cells compared to endothelial differentiated ones. Upregulation of endothelial characteristic genes (Von Willebrand Factor (vWF) and VE Cadherin) was observed in 2D culture. However, PECAM (CD31) was only found to be upregulated after the cells had formed tube-like structures in 3D Matrigel culture. These results indicate that adipose derived CD34+/CD31- cells when cultured in VEGF induced medium, are capable differentiation into endothelial-like lineages. Tube formation of the cells started 3h after seeding the cells on Matrigel and formed more stable and connected network 24 h post seeding in presence of VEGF.

Hybrid Bioprinting of Zonally Stratified Human Articular Cartilage Using Scaffold-Free Tissue Strands as Building Blocks.

The heterogeneous and anisotropic articular cartilage is generally studied as a layered structure of "zones" with unique composition and architecture, which is difficult to recapitulate using current approaches. A novel hybrid bioprinting strategy is presented here to generate zonally stratified cartilage. Scaffold-free tissue strands (TSs) are made of human adipose-derived stem cells (ADSCs) or predifferentiated ADSCs. Cartilage TSs with predifferentiated ADSCs exhibit improved mechanical properties and upregulated expression of cartilage-specific markers at both transcription and protein levels as compared to TSs with ADSCs being differentiated in the form of strands and TSs of nontransfected ADSCs. Using the novel hybrid approach integrating new aspiration-assisted and extrusion-based bioprinting techniques, the bioprinting of zonally stratified cartilage with vertically aligned TSs at the bottom zone and horizontally aligned TSs at the superficial zone is demonstrated, in which collagen fibers are aligned with designated orientation in each zone imitating the anatomical regions and matrix orientation of native articular cartilage. In addition, mechanical testing study reveals a compression modulus of ≈1.1 MPa, which is similar to that of human articular cartilage. The prominent findings highlight the potential of this novel bioprinting approach for building biologically, mechanically, and histologically relevant cartilage for tissue engineering purposes.

Intraoperative Bioprinting: Repairing Tissues and Organs in a Surgical Setting.

3D bioprinting directly into injured sites in a surgical setting, intraoperative bioprinting (IOB), is an effective process, in which the defect information can be rapidly acquired and then repaired via bioprinting on a live subject. In patients needing tissue resection, debridement, traumatic reconstruction, or fracture repair, the ability to scan and bioprint immediately following surgical preparation of the defect site has great potential to improve the precision and efficiency of these procedures. In this opinion article, we provide the reader with current major limitations of IOB from engineering and clinical points of view, as well as possibilities of future translation of bioprinting technologies from bench to bedside, and expound our perspectives in the context of IOB of composite and vascularized tissues.

Cellular Based Strategies for Microvascular Engineering.

Vascularization is a major hurdle in complex tissue and organ engineering. Tissues greater than 200 µm in diameter cannot rely on simple diffusion to obtain nutrients and remove waste. Therefore, an integrated vascular network is required for clinical translation of engineered tissues. Microvessels have been described as <150 µm in diameter, but clinically they are defined as <1 mm. With new advances in super microsurgery, vessels less than 1 mm can be anastomosed to the recipient circulation. However, this technical advancement still relies on the creation of a stable engineered microcirculation that is amenable to surgical manipulation and is readily perfusable. Microvascular engineering lays on the crossroads of microfabrication, microfluidics, and tissue engineering strategies that utilize various cellular constituents. Early research focused on vascularization by co-culture and cellular interactions, with the addition of angiogenic growth factors to promote vascular growth. Since then, multiple strategies have been utilized taking advantage of innovations in additive manufacturing, biomaterials, and cell biology. However, the anatomy and dynamics of native blood vessels has not been consistently replicated. Inconsistent results can be partially attributed to cell sourcing which remains an enigma for microvascular engineering. Variations of endothelial cells, endothelial progenitor cells, and stem cells have all been used for microvascular network fabrication along with various mural cells. As each source offers advantages and disadvantages, there continues to be a lack of consensus. Furthermore, discord may be attributed to incomplete understanding about cell isolation and characterization without considering the microvascular architecture of the desired tissue/organ.

Bioprinting functional tissues.

Despite the numerous lives that have been saved since the first successful procedure in 1954, organ transplant has several shortcomings which prevent it from becoming a more comprehensive solution for medical care than it is today. There is a considerable shortage of organ donors, leading to patient death in many cases. In addition, patients require lifelong immunosuppression to prevent graft rejection postoperatively. With such issues in mind, recent research has focused on possible solutions for the lack of access to donor organs and rejections, with the possibility of using the patient's own cells and tissues for treatment showing enormous potential. Three-dimensional (3D) bioprinting is a rapidly emerging technology, which holds great promise for fabrication of functional tissues and organs. Bioprinting offers the means of utilizing a patient's cells to design and fabricate constructs for replacement of diseased tissues and organs. It enables the precise positioning of cells and biologics in an automated and high throughput manner. Several studies have shown the promise of 3D bioprinting. However, many problems must be overcome before the generation of functional tissues with biologically-relevant scale is possible. Specific focus on the functionality of bioprinted tissues is required prior to clinical translation. In this perspective, this paper discusses the challenges of functionalization of bioprinted tissue under eight dimensions: biomimicry, cell density, vascularization, innervation, heterogeneity, engraftment, mechanics, and tissue-specific function, and strives to inform the reader with directions in bioprinting complex and volumetric tissues. STATEMENT OF SIGNIFICANCE: With thousands of patients dying each year waiting for an organ transplant, bioprinted tissues and organs show the potential to eliminate this ever-increasing organ shortage crisis. However, this potential can only be realized by better understanding the functionality of the organ and developing the ability to translate this to the bioprinting methodologies. Considering the rate at which the field is currently expanding, it is reasonable to expect bioprinting to become an integral component of regenerative medicine. For this purpose, this paper discusses several factors that are critical for printing functional tissues including cell density, vascularization, innervation, heterogeneity, engraftment, mechanics, and tissue-specific function, and inform the reader with future directions in bioprinting complex and volumetric tissues.

Porous tissue strands: avascular building blocks for scalable tissue fabrication.

The scalability of cell aggregates such as spheroids, strands, and rings has been restricted by diffusion of nutrient and oxygen into their core. In this study, we introduce a novel concept in generating tissue building blocks with micropores, which represents an alternative solution for vascularization. Sodium alginate porogens were mixed with human adipose-derived stem cells, and loaded into tubular alginate capsules, followed by de-crosslinking of the capsules. The resultant cellular structure exhibited a porous morphology and formed cell aggregates in the form of strands, called 'porous tissue strands (pTSs).' Three-dimensional reconstructions show that pTSs were able to maintain ∼25% porosity with a high pore interconnectivity (∼85%) for 3 weeks. Owing to the porous structure, pTSs showed up-regulated cell viability and proliferation rate as compared to solid counterparts throughout the culture period. pTSs also demonstrated self-assembly capability through tissue fusion yielding larger-scale patches. In this paper, chondrogenesis and osteogenesis of pTSs were also demonstrated, where the porous microstructure up-regulated both chondrogenic and osteogenic functionalities indicated by cartilage- and bone-specific immunostaining, quantitative biochemical assessment and gene expression. These findings indicated the functionality of pTSs, which possessed controllable porosity and self-assembly capability, and had great potential to be utilized as tissue building blocks in distinct applications such as cartilage and bone regeneration.