Python tooth-inspired fixation device for enhanced rotator cuff repair.

Rotator cuff repair surgeries fail frequently, with 20 to 94% of the 600,000 repairs performed annually in the United States resulting in retearing of the rotator cuff. The most common cause of failure is sutures tearing through tendons at grasping points. To address this issue, we drew inspiration from the specialized teeth of snakes of the Pythonoidea superfamily, which grasp soft tissues without tearing. To apply this nondamaging gripping approach to the surgical repair of tendon, we developed and optimized a python tooth-inspired device as an adjunct to current rotator cuff suture repair and found that it nearly doubled repair strength. Integrated simulations, 3D printing, and ex vivo experiments revealed a relationship between tooth shape and grasping mechanics, enabling optimization of the clinically relevant device that substantially enhances rotator cuff repair by distributing stresses over the attachment footprint. This approach suggests an alternative to traditional suturing paradigms and may reduce the risk of tendon retearing after rotator cuff repair.

The 'bIUreactor': An Open-Source 3D Tissue Research Platform.

We developed the open-source bIUreactor research platform for studying 3D structured tissues. The versatile and modular platform allows a researcher to generate 3D tissues, culture them with oxygenated perfusion, and provide cyclic loading, all in their own lab (in laboratorium) for an all in cost of $8,000 including 3D printer, printing resin, and electronics. We achieved this by applying a design philosophy that leverages 3D printing, open-source software and hardware, and practical techniques to produce the following: 1. perfusible 3D tissues, 2. a bioreactor chamber for tissue culture, 3. a module for applying cyclic compression, 4. a peristaltic pump for providing oxygenated perfusion to 3D tissues, 5. motor control units, and 6. open-source code for running the control units. By making it widely available for researchers to investigate 3D tissue models and easy for them to use, we intend for the bIUreactor to democratize 3D tissue research, therefore increasing the pace and scale of biomedical research discoveries using 3D tissue models.

The Long Road to Develop Custom-built Livers: Current Status of 3D Liver Bioprinting.

Although liver transplantation is the gold-standard therapy for end-stage liver disease, the shortage of suitable organs results in only 25% of waitlisted patients undergoing transplants. Three-dimensional (3D) bioprinting is an emerging technology and a potential solution for personalized medicine applications. This review highlights existing 3D bioprinting technologies of liver tissues, current anatomical and physiological limitations to 3D bioprinting of a whole liver, and recent progress bringing this innovation closer to clinical use. We reviewed updated literature across multiple facets in 3D bioprinting, comparing laser, inkjet, and extrusion-based printing modalities, scaffolded versus scaffold-free systems, development of an oxygenated bioreactor, and challenges in establishing long-term viability of hepatic parenchyma and incorporating structurally and functionally robust vasculature and biliary systems. Advancements in liver organoid models have also increased their complexity and utility for liver disease modeling, pharmacologic testing, and regenerative medicine. Recent developments in 3D bioprinting techniques have improved the speed, anatomical, and physiological accuracy, and viability of 3D-bioprinted liver tissues. Optimization focusing on 3D bioprinting of the vascular system and bile duct has improved both the structural and functional accuracy of these models, which will be critical in the successful expansion of 3D-bioprinted liver tissues toward transplantable organs. With further dedicated research, patients with end-stage liver disease may soon be recipients of customized 3D-bioprinted livers, reducing or eliminating the need for immunosuppressive regimens.

Genetic engineering of porcine endothelial cell lines for evaluation of human-to-pig xenoreactive immune responses.

Xenotransplantation (cross-species transplantation) using genetically-engineered pig organs offers a potential solution to address persistent organ shortage. Current evaluation of porcine genetic modifications is to monitor the nonhuman primate immune response and survival after pig organ xenotransplantation. This measure is an essential step before clinical xenotransplantation trials, but it is time-consuming, costly, and inefficient with many variables. We developed an efficient approach to quickly examine human-to-pig xeno-immune responses in vitro. A porcine endothelial cell was characterized and immortalized for genetic modification. Five genes including GGTA1, CMAH, ß4galNT2, SLA-I α chain, and ß2-microglobulin that are responsible for the production of major xenoantigens (αGal, Neu5Gc, Sda, and SLA-I) were sequentially disrupted in immortalized porcine endothelial cells using CRISPR/Cas9 technology. The elimination of αGal, Neu5Gc, Sda, and SLA-I dramatically reduced the antigenicity of the porcine cells, though the cells still retained their ability to provoke human natural killer cell activation. In summary, evaluation of human immune responses to genetically modified porcine cells in vitro provides an efficient method to identify ideal combinations of genetic modifications for improving pig-to-human compatibility, which should accelerate the application of xenotransplantation to humans.

Comparison of porcine corneal decellularization methods and importance of preserving corneal limbus through decellularization.

BACKGROUND: The aim of this study is to compare the three previously applied, conventional porcine corneal decellularization methods and to demonstrate the importance of preserving the corneal limbus through decellularization. METHODS: Fresh, wild-type (with or without) limbus porcine corneas were decellularized using three different methods, including (i) sodium dodecyl sulfate (SDS), (ii) hypertonic saline (HS), and (iii) N2 gas (NG). Post-treatment evaluation was carried out using histological, residual nuclear material, and ultrastructural analyses. Glycerol was used to help reduce the adverse effects of decellularization. The corneas were preserved for two weeks in cornea storage medium. RESULTS: All three decellularization methods reduced the number of keratocytes at different rates in the stromal tissue. However, all methods, except SDS, resulted in the retention of large numbers of cells and cell fragments. The SDS method (0.1% SDS, 48h) resulted in almost 100% decellularization in corneas without limbus. Low decellularization capacity of the NG method (<50%) could make it unfavorable. Although HS method had a more balanced damage-decellularization ratio, its decellularization capacity was lower than SDS method. Preservation of the corneoscleral limbus could partially prevent structural damage and edema, but it would reduce the decellularization capacity. CONCLUSION: Our results suggest that SDS is a very powerful decellularization method, but it damages the cornea irreversibly. Preserving the corneoscleral limbus reduces the efficiency of decellularization, but also reduces the damage.

Three-Dimensional Biofabrication Models of Endometriosis and the Endometriotic Microenvironment.

Endometriosis occurs when endometrial-like tissue grows outside the uterine cavity, leading to pelvic pain, infertility, and increased risk of ovarian cancer. The present study describes the optimization and characterization of cellular spheroids as building blocks for Kenzan scaffold-free method biofabrication and proof-of-concept models of endometriosis and the endometriotic microenvironment. The spheroid building blocks must be of a specific diameter (~500 µm), compact, round, and smooth to withstand Kenzan biofabrication. Under optimized spheroid conditions for biofabrication, the endometriotic epithelial-like cell line, 12Z, expressed high levels of estrogen-related genes and secreted high amounts of endometriotic inflammatory factors that were independent of TNFα stimulation. Heterotypic spheroids, composed of 12Z and T-HESC, an immortalized endometrial stromal cell line, self-assembled into a biologically relevant pattern, consisting of epithelial cells on the outside of the spheroids and stromal cells in the core. 12Z spheroids were biofabricated into large three-dimensional constructs alone, with HEYA8 spheroids, or as heterotypic spheroids with T-HESC. These three-dimensional biofabricated constructs containing multiple monotypic or heterotypic spheroids represent the first scaffold-free biofabricated in vitro models of endometriosis and the endometriotic microenvironment. These efficient and innovative models will allow us to study the complex interactions of multiple cell types within a biologically relevant microenvironment.

Oxygenation Profiles of Human Blood, Cell Culture Medium, and Water for Perfusion of 3D-Bioprinted Tissues using the FABRICA Bioreactor Platform.

Persistent and saturated oxygen distribution from perfusion media (i.e., blood, or cell culture media) to cells within cell-dense, metabolically-active biofabricated tissues is required to keep them viable. Improper or poor oxygen supply to cells within the tissue bulk severely limits the tissue culturing potential of many bioreactors. We added an oxygenator module to our modular FABRICA bioreactor in order to provide stable oxygenation to biofabricated tissues during culture. In this proof of concept study of an oxygenated and perfused bioreactor, we characterized the oxygenation of water, cell culture medium, and human blood in the FABRICA as functions of augmenting vacuum (air inlet) pressure, perfusion (volumetric flow) rate, and tubing/oxygenator components. The mean oxygen levels for water and cell culture media were 27.7 ± 2.1% and 27.6 ± 4.1%, respectively. The mean oxygen level for human blood was 197.0 ± 90.0 mmHg, with near-physiologic levels achieved with low-permeability PharMed tubing alone (128.0 ± 14.0 mmHg). Hematologic values pre- and post-oxygenation, respectively were (median ± IQR): Red blood cell: 6.0 ± 0.5 (106/µL) and 6.5 ± 0.4 (106/µL); Hemoglobin: 17.5 ± 1.2 g/dL and 19.2 ± 3.0 g/dL; and Hematocrit: 56.7 ± 2.4% and 61.4 ± 7.5%. The relative stability of the hematologic parameters indicates that blood function and thus blood cell integrity were maintained throughout oxygenation. Already a versatile research tool, the now oxygenated FABRICA provides easy-to-implement, in vivo-like perfusion and stable oxygenation culture conditions in vitro semi-independently of one another, which means the bioreactor has the potential to serve as a platform for investigating the behavior of 3D tissue models (regardless of biofabrication method), performing drug toxicity-testing, and testing pharmaceutical efficacy/safety.

Computational fluid dynamic analysis of bioprinted self-supporting perfused tissue models.

Natural tissues are incorporated with vasculature, which is further integrated with a cardiovascular system responsible for driving perfusion of nutrient-rich oxygenated blood through the vasculature to support cell metabolism within most cell-dense tissues. Since scaffold-free biofabricated tissues being developed into clinical implants, research models, and pharmaceutical testing platforms should similarly exhibit perfused tissue-like structures, we generated a generalizable biofabrication method resulting in self-supporting perfused (SSuPer) tissue constructs incorporated with perfusible microchannels and integrated with the modular FABRICA perfusion bioreactor. As proof of concept, we perfused an MLO-A5 osteoblast-based SSuPer tissue in the FABRICA. Although our resulting SSuPer tissue replicated vascularization and perfusion observed in situ, supported its own weight, and stained positively for mineral using Von Kossa staining, our in vitro results indicated that computational fluid dynamics (CFD) should be used to drive future construct design and flow application before further tissue biofabrication and perfusion. We built a CFD model of the SSuPer tissue integrated in the FABRICA and analyzed flow characteristics (net force, pressure distribution, shear stress, and oxygen distribution) through five SSuPer tissue microchannel patterns in two flow directions and at increasing flow rates. Important flow parameters include flow direction, fully developed flow, and tissue microchannel diameters matched and aligned with bioreactor flow channels. We observed that the SSuPer tissue platform is capable of providing direct perfusion to tissue constructs and proper culture conditions (oxygenation, with controllable shear and flow rates), indicating that our approach can be used to biofabricate tissue representing primary tissues and that we can model the system in silico.

Decellularization methods for developing porcine corneal xenografts and future perspectives.

Corneal transplantation is the only option to cure corneal opacities. However, there is an imbalance between supply and demand of corneal tissues in the world. To solve the problem of corneal shortage, corneal xenotransplantation studies have been implemented in the past years using porcine corneas. The corneal xenografts could come from (a) wild-type pigs, (b) genetically engineered pigs, (c) decellularized porcine corneas, and (d) decellularized porcine corneas that are recellularized with human corneal cells, eventually with patients' own cells, as in all type of xenografts. All approaches except, the former would reduce or mitigate recipient immune responses. Although several techniques in decellularization have been reported, there is still no standardized protocol for the complete decellularization of corneal tissue. Herein, we reviewed different decellularization methods for porcine corneas based on the mechanism of action, decellularization efficacy, biocompatibility, and the undesirable effects on corneal ultrastructure. We compared 9 decellularization methods including: (a) sodium dodecyl sulfate, (b) triton x-100, (c) hypertonic saline, (d) human serum with electrophoresis, (e) high hydrostatic pressure, (f) freeze-thaw, (h) nitrogen gas, (h) phospholipase A2 , and (i) glycerol with chemical crosslinking methods. It appears that combined methods could be more useful to perform efficient corneal decellularization.

Scaffold-free bioprinting of mesenchymal stem cells using the Regenova printer: Spheroid characterization and osteogenic differentiation.

Limitations in scaffold material properties, such as sub-optimal degradation time, highlight the need for alternative approaches to engineer de novo tissues. One emerging solution for fabricating tissue constructs is scaffold-free tissue engineering. To facilitate this approach, three-dimensional (3D) bioprinting technology (Regenova Bio 3D Printer) has been developed to construct complex geometric shapes from discrete cellular spheroids without exogenous scaffolds. Optimizing spheroid fabrication and characterizing cellular behavior in the spheroid environment are important first steps prior to printing larger constructs. Here, we characterized spheroids of immortalized mouse bone marrow stromal cells (BMSCs) that were differentiated to the osteogenic lineage. Immortalized BMSCs were seeded in low attachment 96-well plates in various numbers to generate self-aggregated spheroids either under the force of gravity or centrifugation. Cells were cultured in control or osteogenic media for up to 28 days. Spheroid diameter, roundness and smoothness were measured. Cell viability, DNA content and alkaline phosphatase activity were assessed at multiple time points. Additionally, expression of osteogenic markers was determined using real time qPCR. Spheroids formed under gravity with 20 K, 30 K and 40 K cells had average diameters of 498.5 ± 8.3 µm, 580.0 ± 32.9 µm and 639.2 ± 54.0 µm, respectively, while those formed under 300G centrifugation with the same numbers of cells had average diameters of 362.3 ± 3.5 µm, 433.1 ± 6.4 µm and 491.2 ± 8.0 µm. Spheroids formed via centrifugation were superior to those formed by gravity, as evidenced by better roundness and smoothness and double the retention of DNA (cellular) content. Cells in spheroids exhibited a robust osteogenic response to the differentiation medium, including higher mRNA expression of alkaline phosphatase, collagen type I, and osteocalcin than those cultured in control medium, as well as greater alkaline phosphatase activity. The optimal spheroid fabrication technique from this study was to aggregate 40K cells under 150-300G centrifugation. In future investigations, these spheroids will be 3D printed into larger tissue constructs.

Scaffold-free Bioprinting of Mesenchymal Stem Cells with the Regenova Printer: Optimization of Printing Parameters.

The Kenzan bioprinting method provides a high-resolution biofabrication process by facilitating the fusion of submillimeter cell aggregates (spheroids) into larger tissue constructs on a needle array that is removed upon spheroid fusion. Although the method is relatively straightforward in principle, Kenzan method bioprinting relies on a complex 3D bioprinter (Regenova Bio 3D Printer, Cyfuse, K.K., Japan) implementing an advanced vision system to verify the microscopic spheroids' geometry and high-precision mechatronics to aseptically manipulate the spheroids into position. Due to the complexity of the operation, the need for aseptic conditions, and the size of the spheroids, proficiency with the Regenova Bio 3D Printer and the Kenzan method requires development of best practices and troubleshooting techniques to ensure a robust print and minimize the use of resources. In addition, managing the construct post-bioprinting both in culture and for surgical implantation requires careful consideration and workflow design. Here, we describe methods for generating a competent tissue construct and optimizing the bioprinting process. Optimization resulted in a 4-fold reduction in print times, a 20-fold reduction in the use of bioprinting nozzles, and more robust constructs. The results and procedures described herein will have potential applications for tissue engineering, research, and clinical uses in the future.

The potential role of 3D-bioprinting in xenotransplantation.

PURPOSE OF REVIEW: To review the impact of a new technology, 3D-bioprinting, in xenotransplantation research. RECENT FINDINGS: Genetically engineered pigs, beginning with human (h) CD55-transgenic and Gal-knockout pigs, have improved the outcomes of xenotransplantation research. Today, there are more than 30 different genetically engineered pigs either expressing human gene(s) or lacking pig gene(s). CRIPSR/cas9 technology has facilitated the production of multigene pigs (up to nine genes in a single pig), which lack multiple pig xenoantigens, and express human transgenes, such as hCD46, hCD55, hThrombomodulin, hCD39, etc. Although recent studies in nonhuman primates (NHPs) have demonstrated prolonged survival after life-supporting pig kidney, heart, and islet xenotransplantation, researchers have difficulty determining the best genetic combination to test in NHPs because of a potential greater than 100 000 genetic combinations. 3D-bioprinting of genetically engineered pig cells: is superior to 2D in-vitro testing, enables organ-specific testing, helps to understand differences in immunogenicity between organs, and is faster and cheaper than testing in NHPs. Moreover, 3D-bioprinted cells can be continuously perfused in a bioreactor, controlling for all variables, except the studied variable. SUMMARY: 3D-bioprinting can help in the study of the impact of specific genes (human or pig) in xenotransplantation in a rapid, inexpensive, and reliable way.

FABRICA: A Bioreactor Platform for Printing, Perfusing, Observing, & Stimulating 3D Tissues.

We are introducing the FABRICA, a bioprinter-agnostic 3D-printed bioreactor platform designed for 3D-bioprinted tissue construct culture, perfusion, observation, and analysis. The computer-designed FABRICA was 3D-printed with biocompatible material and used for two studies: (1) Flow Profile Study: perfused 5 different media through a synthetic 3D-bioprinted construct and ultrasonically analyzed the flow profile at increasing volumetric flow rates (VFR); (2) Construct Perfusion Study: perfused a 3D-bioprinted tissue construct for a week and compared histologically with a non-perfused control. For the flow profile study, construct VFR increased with increasing pump VFR. Water and other media increased VFR significantly while human and pig blood showed shallow increases. For the construct perfusion study, we confirmed more viable cells in perfused 3D-bioprinted tissue compared to control. The FABRICA can be used to visualize constructs during 3D-bioprinting, incubation, and to control and ultrasonically analyze perfusion, aseptically in real-time, making the FABRICA tunable for different tissues.

Tunability of collagen matrix mechanical properties via multiple modes of mineralization.

Functionally graded, mineralized collagen tissues exist at soft-to-hard material attachments throughout the body. However, the details of how collagen and hydroxyapatite mineral (HA) interact are not fully understood, hampering efforts to develop tissue-engineered constructs that can assist with repair of injuries at the attachments of soft tissues to bone. In this study, spatial control of mineralization was achieved in collagen matrices using simulated body fluids (SBFs). Based upon previous observations of poor bonding between reconstituted collagen and HA deposited using SBF, we hypothesized that mineralizing collagen in the presence of fetuin (which inhibits surface mineralization) would lead to more mineral deposition within the scaffold and therefore a greater increase in stiffness and toughness compared with collagen mineralized without fetuin. We tested this hypothesis through integrated synthesis, mechanical testing and modelling of graded, mineralized reconstituted collagen constructs. Results supported the hypothesis, and further suggested that mineralization on the interior of reconstituted collagen constructs, as promoted by fetuin, led to superior bonding between HA and collagen. The results provide us guidance for the development of mineralized collagen scaffolds, with implications for bone and tendon-to-bone tissue engineering.

Dissipation of sulfamethoxazole and trimethoprim antibiotics from manure-amended soils.

In this study, the dissipation of two antibiotics, sulfamethoxazole (SMX) and trimethoprim (TRM), in three soils under both aerobic and anaerobic conditions are evaluated. Under aerobic conditions, SMX dissipated rapidly through biodegradation but TRM was more persistent. Within the first 20 days in biologically active soils, >50% of the SMX was lost from the clay loam and loamy sand soils, and >80% loss was noted in the loam soil. Anaerobic dissipation of both compounds was more rapid than aerobic dissipation. The addition of manure to the soil only slightly increased the initial dissipation rate of the two compounds. Little effect was found on glucose mineralisation in soil following the addition of SMX and TRM, even as mixtures at high concentrations.

Tissue-engineering strategies for the tendon/ligament-to-bone insertion.

Injuries to connective tissues are painful and disabling and result in costly medical expenses. These injuries often require reattachment of an unmineralized connective tissue to bone. The uninjured tendon/ligament-to-bone insertion (enthesis) is a functionally graded material that exhibits a gradual transition from soft tissue (i.e., tendon or ligament) to hard tissue (i.e., mineralized bone) through a fibrocartilaginous transition region. This transition is believed to facilitate force transmission between the two dissimilar tissues by ameliorating potentially damaging interfacial stress concentrations. The transition region is impaired or lost upon tendon/ligament injury and is not regenerated following surgical repair or natural healing, exposing the tissue to risk of reinjury. The need to regenerate a robust tendon-to-bone insertion has led a number of tissue engineering repair strategies. This review treats the tendon-to-bone insertion site as a tissue structure whose primary role is mechanical and discusses current and emerging strategies for engineering the tendon/ligament-to-bone insertion in this context. The focus lies on strategies for producing mechanical structures that can guide and subsequently sustain a graded tissue structure and the associated cell populations.

Poor efficacy of herbicides in biochar-amended soils as affected by their chemistry and mode of action.

We evaluated wheat straw biochar produced at 450°C for its ability to influence bioavailability and persistence of two commonly used herbicides (atrazine and trifluralin) with different modes of action (photosynthesis versus root tip mitosis inhibitors) in two contrasting soils. The biochar was added to soils at 0%, 0.5% and 1.0% (w/w) and the herbicides were applied to those soil-biochar mixes at nil, half, full, two times, and four times, the recommended dosage (H(4)). Annual ryegrass (Lolium rigidum) was grown in biochar amended soils for 1 month. Biochar had a positive impact on ryegrass survival rate and above-ground biomass at most of the application rates, and particularly at H(4). Within any given biochar treatment, increasing herbicide application decreased the survival rate and fresh weight of above-ground biomass. Biomass production across the biochar treatment gradient significantly differed (p<0.01) and was more pronounced in the case of atrazine than trifluralin. For example, the dose-response analysis showed that in the presence of 1% biochar in soil, the value of GR(50) (i.e. the dose required to reduce weed biomass by 50%) for atrazine increased by 3.5 times, whereas it increased only by a factor of 1.6 in the case of trifluralin. The combination of the chemical properties and the mode of action governed the extent of biochar-induced reduction in bioavailability of herbicides. The greater biomass of ryegrass in the soil containing the highest biochar (despite having the highest herbicide residues) demonstrates decreased bioavailability of the chemicals caused by the wheat straw biochar. This work clearly demonstrates decreased efficacy of herbicides in biochar amended soils. The role played by herbicide chemistry and mode of action will have major implications in choosing the appropriate application rates for biochar amended soils.

Fibrocartilage tissue engineering: the role of the stress environment on cell morphology and matrix expression.

Although much is known about the effects of uniaxial mechanical loading on fibrocartilage development, the stress fields to which fibrocartilaginous regions are subjected to during development are mutiaxial. That fibrocartilage develops at tendon-to-bone attachments and in compressive regions of tendons is well established. However, the three-dimensional (3D) nature of the stresses needed for the development of fibrocartilage is not known. Here, we developed and applied an in vitro system to determine whether fibrocartilage can develop under a state of periodic hydrostatic tension in which only a single principal component of stress is compressive. This question is vital to efforts to mechanically guide morphogenesis and matrix expression in engineered tissue replacements. Mesenchymal stromal cells in a 3D culture were exposed to compressive and tensile stresses as a result of an external tensile hydrostatic stress field. The stress field was characterized through mechanical modeling. Tensile cyclic stresses promoted spindle-shaped cells, upregulation of scleraxis and type one collagen, and cell alignment with the direction of tension. Cells experiencing a single compressive stress component exhibited rounded cell morphology and random cell orientation. No difference in mRNA expression of the genes Sox9 and aggrecan was observed when comparing tensile and compressive regions unless the medium was supplemented with the chondrogenic factor transforming growth factor beta3. In that case, Sox9 was upregulated under static loading conditions and aggrecan was upregulated under cyclic loading conditions. In conclusion, the fibrous component of fibrocartilage could be generated using only mechanical cues, but generation of the cartilaginous component of fibrocartilage required biologic factors in addition to mechanical cues. These studies support the hypothesis that the 3D stress environment influences cell activity and gene expression in fibrocartilage development.

Decreased fibroblast and increased osteoblast adhesion on nanostructured NaOH-etched PLGA scaffolds.

To facilitate locomotion and support the body, the skeleton relies on the transmission of forces between muscles and bones through complex junctions called entheses. The varying mechanical and biological properties of the enthesis make healing this avascular tissue difficult; hence the need for an engineered alternative. Cells in situ interact with their environment on the nano-scale which suggests that engineered approaches to enthesis regeneration should include such biologically-inspired nano-scale surface features. The present in vitro study investigated the effects of etching poly-lactic-co-glycolic acid (PLGA) scaffolds to produce nano-topography on the adhesion of fibroblasts and osteoblasts, two integral enthesis cell types. Nano-topography was produced on PLGA by etching the scaffolds in NaOH. Results showed that etching PLGA with NaOH to create nano-scale surface features decreased fibroblast adhesion while it increased osteoblast adhesion; criteria critical for the spatial control of osteoblast and fibroblast adhesion for a successful enthesis tissue engineering material. Thus, the results of this study showed for the first time collective evidence that PLGA can be either treated with NaOH or not on ends of an enthesis tissue engineering construct to spatially increase osteoblast and fibroblast adhesion, respectively.