Reconstructing Curves: A Bottom-Up Approach toward Adipose Tissue Regeneration with Recombinant Biomaterials.

The potential of recombinant materials in the field of adipose tissue engineering (ATE) is investigated using a bottom-up tissue engineering (TE) approach. This study explores the synthesis of different photo-crosslinkable gelatin derivatives, including both natural and recombinant materials, with a particular emphasis on chain growth and step growth polymerization. Gelatin type B (Gel-B) and a recombinant collagen peptide (RCPhC1) are used as starting materials. The gel fraction and mass swelling properties of 2D hydrogel films are evaluated, revealing high gel fractions exceeding 94% and high mass swelling ratios >15. In vitro experiments with encapsulated adipose-derived stem cells (ASCs) indicate viable cells (>85%) throughout the experiment with the RCPhC1-based hydrogels showing a higher number of stretched ASCs. Triglyceride assays show the enhanced differentiation potential of RCPhC1 materials. Moreover, the secretome analysis reveal the production of adipose tissue-specific proteins including adiponectin, adipsin, lipocalin-2/NGAL, and PAL-1. RCPhC1-based materials exhibit higher levels of adiponectin and adipsin production, indicating successful differentiation into the adipogenic lineage. Overall, this study highlights the potential of recombinant materials for ATE applications, providing insights into their physico-chemical properties, mechanical strength, and cellular interactions.

Powdered Cross-Linked Gelatin Methacryloyl as an Injectable Hydrogel for Adipose Tissue Engineering.

The tissue engineering field is currently advancing towards minimally invasive procedures to reconstruct soft tissue defects. In this regard, injectable hydrogels are viewed as excellent scaffold candidates to support and promote the growth of encapsulated cells. Cross-linked gelatin methacryloyl (GelMA) gels have received substantial attention due to their extracellular matrix-mimicking properties. In particular, GelMA microgels were recently identified as interesting scaffold materials since the pores in between the microgel particles allow good cell movement and nutrient diffusion. The current work reports on a novel microgel preparation procedure in which a bulk GelMA hydrogel is ground into powder particles. These particles can be easily transformed into a microgel by swelling them in a suitable solvent. The rheological properties of the microgel are independent of the particle size and remain stable at body temperature, with only a minor reversible reduction in elastic modulus correlated to the unfolding of physical cross-links at elevated temperatures. Salts reduce the elastic modulus of the microgel network due to a deswelling of the particles, in addition to triple helix denaturation. The microgels are suited for clinical use, as proven by their excellent cytocompatibility. The latter is confirmed by the superior proliferation of encapsulated adipose tissue-derived stem cells in the microgel compared to the bulk hydrogel. Moreover, microgels made from the smallest particles are easily injected through a 20G needle, allowing a minimally invasive delivery. Hence, the current work reveals that powdered cross-linked GelMA is an excellent candidate to serve as an injectable hydrogel for adipose tissue engineering.

Vascularized adipose tissue engineering: moving towards soft tissue reconstruction.

Soft tissue defects are a common clinical challenge mostly caused by trauma, congenital anomalies and oncological surgery. Current soft tissue reconstruction options include synthetic materials (fillers and implants) and autologous adipose tissue transplantation through flap surgery and/or lipotransfer. Both reconstructive options hold important disadvantages to which vascularized adipose tissue engineering (VATE) strategies could offer solutions. In this review, we first summarized pivotal characteristics of functional adipose tissue such as the structure, function, cell types, development and extracellular matrix (ECM). Next, we discussed relevant cell sources and how they are applied in different state-of-the-art VATE techniques. Herein, biomaterial scaffolds and hydrogels, ECMs, spheroids, organoids, cell sheets, three dimensional printing and microfluidics are overviewed. Also, we included extracellular vesicles and emphasized their potential role in VATE. Lastly, current challenges and future perspectives in VATE are pointed out to help to pave the road towards clinical applications.

Flexor tendon repair using a reinforced tubular, medicated electrospun construct.

A reinforced tubular, medicated electrospun construct was developed for deep flexor tendon repair. This construct combines mechanical strength with the release of anti-inflammatory and anti-adhesion drugs. In this study, the reinforced construct was evaluated using a rabbit model. It was compared to its components (a tubular, medicated electrospun polymer without reinforcement and a tubular braid as such) on the one hand to a modified Kessler suture as a control group. Forty New Zealand rabbits were randomly divided into two groups. Surgery was performed in the second and fourth deep flexor tendons of one hind paw of the rabbits in the two groups using four repair techniques. Biomechanical tensile testing and macroscopic and histological evaluations were performed at 3 and 8 weeks postoperatively. A two-way analysis of variance with pairwise comparisons revealed that the three experimental surgical techniques (a reinforced tubular medicated electrospun construct, tubular-medicated construct, and tubular braid as such) showed similar strength as that of a modified Kessler suture repair, which was characterized by a mean load at ultimate failure of 19.85 N (standard deviation [SD] 5.29 N) at 3 weeks and 18.15 N (SD 8.01 N) at 8 weeks. Macroscopically, a significantly different adhesion pattern was observed at the suture knots, either centrally or peripherally, depending on the technique. Histologically, a qualitative assessment showed good to excellent repair at the tendon repair site, irrespective of the applied technique. This study demonstrates that mechanical and biological repair strategies for flexor tendon repair can be successfully combined.

Potential of poly(alkylene terephthalate)s to control endothelial cell adhesion and viability.

Poly(ethylene terephthalate) (PET) is known for its various useful characteristics, including its applicability in cardiovascular applications, more precisely as synthetic bypass grafts for large diameter (≥ 6 mm) blood vessels. Although it is widely used, PET is not an optimal material as it is not interactive with endothelial cells, which is required for bypasses to form a complete endothelium. Therefore, in this study, poly(alkylene terephthalate)s (PATs) have been studied. They were synthesized via a single-step solution polycondensation reaction, which requires mild reaction conditions and avoids the use of a catalyst or additives like heat stabilizers. A homologous series was realized in which the alkyl chain length varied from 5 to 12 methylene groups (n = 5-12). Molar masses up to 28,000 g/mol were obtained, while various odd-even trends were observed with modulated differential scanning calorimetry (mDSC) and rapid heat-cool calorimetry (RHC) to access the thermal properties within the homologous series. The synthesized PATs have been subjected to in vitro cell viability assays using Human Umbilical Vein Endothelial Cells (HUVECs) and Human Dermal Microvascular Endothelial Cells (HDMECs). The results showed that HUVECs adhere and proliferate most pronounced onto PAT(n=9) surfaces, which could be attributed to the surface roughness and morphology as determined by atomic force microscopy (AFM) (i.e. Rq = 204.7 nm). HDMECs were investigated in the context of small diameter vessels and showed superior adhesion and proliferation after seeding onto PAT(n=6) substrates. These preliminary results already pave the way towards the use of PAT materials as substrates to support endothelial cell adhesion and growth. Indeed, as superior endothelial cell interactivity compared to PET was observed, time-consuming and costly surface modifications of PET grafts could be avoided by exploiting this novel material class.

Photo-Crosslinked Gelatin-Based Hydrogel Films to Support Wound Healing.

Gelatin is used widely in the biomedical field, among other for wound healing. Given its upper critical solution temperature, crosslinking is required. To this end, gelatin is chemically modified with different photo-crosslinkable moieties with low (32-34%) and high (63-65%) degree of substitution (DS): gelatin-methacrylamide (gel-MA) and gelatin-acrylamide (gel-AA) and gelatin-pentenamide (gel-PE). Next to the more researched gel-MA, it is especially interesting and novel to compare with other gelatin-derived compounds for the application of wound healing. An additional comparison is made with commercial dressings. The DS is directly proportional to the mechanical characteristics and inversely proportional to the swelling capacity. Gel-PE shows weaker mechanical properties (G' < 15 kPa) than gel-AA and gel-MA (G' < 39 and 45 kPa, respectively). All derivatives are predominantly elastic (recovery indices of 89-94%). Gel-AA and gel-MA show excellent biocompatibility, whereas gel-PE shows a significantly lower initial biocompatibility, evolving positively toward day 7. Overall, gel-MA shows to have the most potential to be applied as wound dressing. Future blending with gel-AA to improve the curing kinetics can lead to dressings able to compete with current commercial dressings.

Toward Adipose Tissue Engineering Using Thiol-Norbornene Photo-Crosslinkable Gelatin Hydrogels.

Nowadays, breast implants, lipofilling, and microsurgical free tissue transfer are the most often applied procedures to repair soft tissue defects resulting from mastectomies/lumpectomies following breast cancer. Due to the drawbacks and limitations associated with these conventional clinical practices, there is a need for alternative reconstructive strategies. The development of biomimetic materials able to promote cell proliferation and adipogenic differentiation has gained increasing attention in the context of adipose reconstructive purposes. Herein, thiol-norbornene crosslinkable gelatin-based materials were developed and benchmarked to the current commonly applied methacryloyl-modified gelatin (GelMA) with different degrees of substitutions focussing on bottom-up tissue engineering. The developed hydrogels resulted in similar gel fractions, swelling, and in vitro biodegradation properties compared to the benchmark materials. Furthermore, the thiol-ene hydrogels exhibited mechanical properties closer to those of native fatty tissue compared to GelMA. The mechanical cues of the equimolar GelNB DS55% + GelSH DS75% composition resulted not only in similar biocompatibility but also, more importantly, in superior differentiation of the encapsulated cells into the adipogenic lineage, as compared to GelMA. It can be concluded that the photo-crosslinkable thiol-ene systems offer a promising strategy toward adipose tissue engineering through cell encapsulation compared to the benchmark GelMA.

Equine Tenocyte Seeding on Gelatin Hydrogels Improves Elongated Morphology.

(1) Background: Tendinopathy is a common injury in both human and equine athletes. Representative in vitro models are mandatory to facilitate translation of fundamental research into successful clinical treatments. Natural biomaterials like gelatin provide favorable cell binding characteristics and are easily modifiable. In this study, methacrylated gelatin (gel-MA) and norbornene-functionalized gelatin (gel-NB), crosslinked with 1,4-dithiotreitol (DTT) or thiolated gelatin (gel-SH) were compared. (2) Methods: The physicochemical properties (1H-NMR spectroscopy, gel fraction, swelling ratio, and storage modulus) and equine tenocyte characteristics (proliferation, viability, and morphology) of four different hydrogels (gel-MA, gel-NB85/DTT, gel-NB55/DTT, and gel-NB85/SH75) were evaluated. Cellular functionality was analyzed using fluorescence microscopy (viability assay and focal adhesion staining). (3) Results: The thiol-ene based hydrogels showed a significantly lower gel fraction/storage modulus and a higher swelling ratio compared to gel-MA. Significantly less tenocytes were observed on gel-MA discs at 14 days compared to gel-NB85/DTT, gel-NB55/DTT and gel-NB85/SH75. At 7 and 14 days, the characteristic elongated morphology of tenocytes was significantly more pronounced on gel-NB85/DTT and gel-NB55/DTT in contrast to TCP and gel-MA. (4) Conclusions: Thiol-ene crosslinked gelatins exploiting DTT as a crosslinker are the preferred biomaterials to support the culture of tenocytes. Follow-up experiments will evaluate these biomaterials in more complex models.

Design and development of a reinforced tubular electrospun construct for the repair of ruptures of deep flexor tendons.

This research aims at developing a more potent solution for deep flexor tendon repair by combining a mechanical and biological approach. A reinforced, multi-layered electrospun tubular construct is developed, composed of three layers: an inner electrospun layer containing an anti-inflammatory component (Naproxen), a middle layer of braided monofilament as reinforcement and an outer electrospun layer containing an anti-adhesion component (hyaluronic acid, HA). In a first step, a novel acrylate endcapped urethane-based precursor (AUP) is developed and characterized by measuring molar mass, acrylate content and thermo-stability. The AUP material is benchmarked against commercially available poly(ε-caprolactone) (PCL). Next, the materials are processed into multi-layered, tubular constructs with bio-active components (Naproxen and HA) using electrospinning. In vitro assays using human fibroblasts show that incorporation of the bio-active components is successful and not-cytotoxic. Moreover, tensile testing using ex vivo sheep tendons prove that the developed multi-layered constructs fulfill the required strength for tendon repair (i.e. 2.79-3.98 MPa), with an ultimate strength of 8.56 ±â€¯1.92 MPa and 8.36 ±â€¯0.57 MPa for PCL and AUP/PCL constructs respectively. In conclusion, by combining a mechanical approach (improved mechanical properties) with the incorporation of bio-active compounds (biological approach), this solution shows its potential for application in deep flexor tendon repair.

Thiol-norbornene gelatin hydrogels: influence of thiolated crosslinker on network properties and high definition 3D printing.

Photocrosslinkable gelatin hydrogels are excellent bioinks or biomaterial ink components to serve biofabrication applications. Especially the widely investigated gelatin-methacroyl (gel-MA) hydrogels hold an impressive track record. However, over the past decade, increasing attention is being paid to thiol-ene photo-click chemistry to obtain hydrogel networks benefitting from a faster reactivity (i.e. seconds vs minutes) along with superior biocompatibility and processability. In order to exploit this photo-click chemistry, often an ene-functionality (e.g. norbornene) is introduced onto gelatin followed by crosslinking in the presence of a multifunctional thiol (e.g. dithiothreitol). To date, very limited research has been performed on the influence of the applied thiolated crosslinker on the final hydrogel properties. Therefore, the present work assesses the influence of different thiolated crosslinkers on the crosslinking kinetics, mechanical properties and biological performance of the hydrogels upon encapsulation of primary adipose tissue-derived stem cells which indicated a cell viability exceeding 70%. Furthermore, the different formulations were processed using two-photon polymerization which indicated, in addition to differences in processing window and swelling ratio, a previously unreported phenomenon. At high intensities (i.e. ⩾150 mW), the laser results in cleavage of the gelatin backbone even in the absence of distinct photo-cleavable functionalities. This can have potential to introduce channels or softer regions in gels to result in zones characterized by different degradation speeds or the formation of blood vessels. Consequently, the present study can be used to provide guidance towards tailoring the thiol-ene system towards the desired applications.

High-Resolution 3D Bioprinting of Photo-Cross-linkable Recombinant Collagen to Serve Tissue Engineering Applications.

Various biopolymers, including gelatin, have already been applied to serve a plethora of tissue engineering purposes. However, substantial concerns have arisen related to the safety and the reproducibility of these materials due to their animal origin and the risk associated with pathogen transmission as well as batch-to-batch variations. Therefore, researchers have been focusing their attention toward recombinant materials that can be produced in a laboratory with full reproducibility and can be designed according to specific needs (e.g., by introducing additional RGD sequences). In the present study, a recombinant protein based on collagen type I (RCPhC1) was functionalized with photo-cross-linkable methacrylamide (RCPhC1-MA), norbornene (RCPhC1-NB), or thiol (RCPhC1-SH) functionalities to enable high-resolution 3D printing via two-photon polymerization (2PP). The results indicated a clear difference in 2PP processing capabilities between the chain-growth-polymerized RCPhC1-MA and the step-growth-polymerized RCPhC1-NB/SH. More specifically, reduced swelling-related deformations resulting in a superior CAD-CAM mimicry were obtained for the RCPhC1-NB/SH hydrogels. In addition, RCPhC1-NB/SH allowed the processing of the material in the presence of adipose tissue-derived stem cells that survived the encapsulation process and also were able to proliferate when embedded in the printed structures. As a consequence, it is the first time that successful HD bioprinting with cell encapsulation is reported for recombinant hydrogel bioinks. Therefore, these results can be a stepping stone toward various tissue engineering applications.

Evaluation of 3D Printed Gelatin-Based Scaffolds with Varying Pore Size for MSC-Based Adipose Tissue Engineering.

Adipose tissue engineering aims to provide solutions to patients who require tissue reconstruction following mastectomies or other soft tissue trauma. Mesenchymal stromal cells (MSCs) robustly differentiate into the adipogenic lineage and are attractive candidates for adipose tissue engineering. This work investigates whether pore size modulates adipogenic differentiation of MSCs toward identifying optimal scaffold pore size and whether pore size modulates spatial infiltration of adipogenically differentiated cells. To assess this, extrusion-based 3D printing is used to fabricate photo-crosslinkable gelatin-based scaffolds with pore sizes in the range of 200-600 µm. The adipogenic differentiation of MSCs seeded onto these scaffolds is evaluated and robust lipid droplet formation is observed across all scaffold groups as early as after day 6 of culture. Expression of adipogenic genes on scaffolds increases significantly over time, compared to TCP controls. Furthermore, it is found that the spatial distribution of cells is dependent on the scaffold pore size, with larger pores leading to a more uniform spatial distribution of adipogenically differentiated cells. Overall, these data provide first insights into the role of scaffold pore size on MSC-based adipogenic differentiation and contribute toward the rational design of biomaterials for adipose tissue engineering in 3D volumetric spaces.

Extrusion-based 3D printing of photo-crosslinkable gelatin and κ-carrageenan hydrogel blends for adipose tissue regeneration.

Current soft tissue repair techniques for women with breast cancer remain associated with several drawbacks including surgical complications and a high resorption rate for lipofilling techniques. Hence, the need to develop improved adipose tissue reconstruction strategies. Additive manufacturing can be a promising tool towards the development of patient-specific scaffolds which are able to support adipose tissue engineering. In the present work, scaffolds composed of both methacrylamide-modified gelatin (Gel-MA) and methacrylated κ-carrageenan (Car-MA), i.e. hydrogel blends, were developed using extrusion-based 3D printing in order to establish a close resemblance to the native extracellular matrix. The hydrogel blends were benchmarked to scaffolds constituting of only Gel-MA. Our results indicate that both types of scaffolds remain stable over time (21 days), are able to absorb large amounts of water and exhibit mechanical properties comparable to those of native breast tissue (2 kPa). Furthermore, a similar cell viability (> 90%) and proliferation rate after 14 days was obtained for adipose tissue-derived stem cells (ASCs) upon seeding onto both types of scaffolds. Additionally, the ASCs were able to differentiate into the adipogenic lineage on the hydrogel blend scaffolds, although their differentiation potential was lower compared to that of ASCs seeded onto the Gel-MA scaffolds.

Additive manufacturing of photo-crosslinked gelatin scaffolds for adipose tissue engineering.

There exists a clear clinical need for adipose tissue reconstruction strategies to repair soft tissue defects which outperform the currently available approaches. In this respect, additive manufacturing has shown to be a promising alternative for the development of larger constructs able to support adipose tissue engineering. In the present work, a thiol-ene photo-click crosslinkable gelatin hydrogel was developed which allowed extrusion-based additive manufacturing into porous scaffolds. To this end, norbornene-functionalized gelatin (Gel-NB) was combined with thiolated gelatin (Gel-SH). The application of a macromolecular gelatin-based thiolated crosslinker holds several advantages over conventional crosslinkers including cell-interactivity, less chance at phase separation between scaffold material and crosslinker and the formation of a more homogeneous network. Throughout the paper, these photo-click scaffolds were benchmarked to the conventional methacrylamide-modified gelatin (Gel-MA). The results indicated that stable scaffolds could be realized which were further characterized physico-chemically by performing swelling, mechanical and in vitro biodegradability assays. Furthermore, the seeded adipose tissue-derived stem cells (ASCs) remained viable (>90%) up to 14 days and were able to proliferate. In addition, the cells could be differentiated into the adipogenic lineage on the photo-click crosslinked scaffolds, thereby performing better than the cells supported by the frequently reported Gel-MA scaffolds. As a result, the developed photo-click crosslinked scaffolds can be considered a promising candidate towards adipose tissue engineering and a valuable alternative for the omnipresent Gel-MA. STATEMENT OF SIGNIFICANCE: The field of adipose tissue engineering has emerged as a promising strategy to repair soft tissue defects. Herein, Gel-NB/Gel-SH gelatin-based hydrogel scaffolds were produced using extrusion-based additive manufacturing. Using a cell-interactive, thiolated gelatin crosslinker, a homogeneous network was formed and the risk of phase separation between norbornene-modified gelatin and macromolecular crosslinkers was reduced. UV-induced crosslinking of these materials is based on step growth polymerization which requires less free radicals to enable polymerization. Our results demonstrated the potential of the developed scaffolds, due to their favourable physico-chemical characteristics as well as their adipogenic differentiation potential when benchmarked to Gel-MA scaffolds. Hence, the hydrogels could be of great interest towards future development of adipose tissue constructs and tissue engineering in general.

Effect of decompressive laparotomy on organ function in patients with abdominal compartment syndrome: a systematic review and meta-analysis.

BACKGROUND: Decompressive laparotomy has been advised as potential treatment for abdominal compartment syndrome (ACS) when medical management fails; yet, the effect on parameters of organ function differs markedly in the published literature. In this study, we sought to investigate the effect of decompressive laparotomy on intra-abdominal pressure and organ function in critically ill adult and pediatric patients with ACS, specifically focusing on hemodynamic, respiratory, and kidney function and outcome. METHODS: A systematic review and meta-analysis of the literature was performed. Articles reporting data on intra-abdominal pressure (IAP), hemodynamic (mean arterial pressures [MAP], central venous pressure [CVP], cardiac index [CI], heart rate [HR], systemic vascular resistance index [SVRI] and/or pulmonary capillary wedge pressure [PCWP]), respiratory (positive end-expiratory pressure [PEEP], peak inspiratory pressure [PIP] and/or ratio of partial pressure arterial oxygen and fraction of inspired oxygen [P/F ratio]), and/or urinary output (UO) following decompressive laparotomy were analyzed. RESULTS: A total of 15 articles were included; 3 included children only (aged 18 years or younger). Of the 286 patients who were included, 49.7% had primary ACS. The baseline mean IAP in adults decreased with an average of 18.2 ± 6.5 mmHg following decompression, from 31.7 ± 6.4 mmHg to 13.5 ± 3.0 mmHg. There was a decrease in HR (12.2 ± 9.5 beats/min; p = 0.04), CVP (4.6 ± 2.3 mmHg; p = 0.022), PCWP (5.8 ± 2.3 mmHg; p = 0.029), and PIP (10.1 ± 3.9 cmH2O; p < 0.001) and a mean increase in P/F ratio (70.4 ± 49.4; p < 0.001) and UO (95.3 ± 105.3 ml/h; p < 0.001). In children, there was a significant increase in MAP (20.0 ± 2.3 mmHg; p = 0.006), P/F ratio (238.2; p < 0.001), and UO (2.88 ± 0.64 ml/kg/h; p < 0.001) and a decrease in CVP (7 mmHg; p = 0.016) and PIP (9.9 cmH2O; p = 0.002). The overall mortality rate was 49.7% in adults and 60.8% in children following decompressive laparotomy. CONCLUSIONS: Decompressive laparotomy resulted in a significantly lower IAP and had beneficial effects on hemodynamic, respiratory, and renal parameters. Mortality after decompressive laparotomy remains high in both adults and children.