Farnesol prevents aging-related muscle weakness in mice through enhanced farnesylation of Parkin-interacting substrate.

Peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) is a master regulator of mitochondrial biogenesis. Reduced PGC-1α abundance is linked to skeletal muscle weakness in aging or pathological conditions, such as neurodegenerative diseases and diabetes; thus, elevating PGC-1α abundance might be a promising strategy to treat muscle aging. Here, we performed high-throughput screening and identified a natural compound, farnesol, as a potent inducer of PGC-1α. Farnesol administration enhanced oxidative muscle capacity and muscle strength, leading to metabolic rejuvenation in aged mice. Moreover, farnesol treatment accelerated the recovery of muscle injury associated with enhanced muscle stem cell function. The protein expression of Parkin-interacting substrate (PARIS/Zfp746), a transcriptional repressor of PGC-1α, was elevated in aged muscles, likely contributing to PGC-1α reduction. The beneficial effect of farnesol on aged muscle was mediated through enhanced PARIS farnesylation, thereby relieving PARIS-mediated PGC-1α suppression. Furthermore, short-term exercise increased PARIS farnesylation in the muscles of young and aged mice, whereas long-term exercise decreased PARIS expression in the muscles of aged mice, leading to the elevation of PGC-1α. Collectively, the current study demonstrated that the PARIS-PGC-1α pathway is linked to muscle aging and that farnesol treatment can restore muscle functionality in aged mice through increased farnesylation of PARIS.

Laparoscopic renal surgery using multi degree-of-freedom articulating laparoscopic instruments in a porcine model.

PURPOSE: We evaluated the performance of a new multi-degree-of-freedom articulating laparoscopic instrument, ArtiSential, and compared it with that of a straight-shaped instrument and the da Vinci surgical system, in renal surgery using porcine model. MATERIALS AND METHODS: Nine female Yorkshire pigs were equally divided into three groups. The three groups were compared at each surgical step in terms of objective and subjective parameters. RESULTS: The median operative times for renal pedicle clamping and ureter dissection were significantly shorter in ArtiSential group than robotic group (1.3 min vs. 4.7 min, p=0.002; 8.1 min vs. 11.1 min, p=0.015). The median operative time for bladder repair was significantly longer in ArtiSential group than robotic and straight-shaped groups (17.9 min vs. 5.5 min, p=0.002; 17.9 min vs. 9.3 min, p=0.026). There were no significant differences among groups in terms of blood loss or intraoperative complications. ArtiSential device was less useable for renorrhaphy (p=0.009) and bladder repair (p=0.002) compared to the robotic system. ArtiSential group was less accurate than robotic group in terms of tumor resection, renorrhaphy, and bladder repair. During ureter dissection, bladder cuff excision, and bladder repair, the surgeon experienced greater wrist discomfort but lesser back discomfort in ArtiSential group than robotic group. CONCLUSIONS: For most steps, ArtiSential performed as well as robotic and straight-shaped instruments. The development of specialized surgical techniques for ArtiSential will maximize the advantages of these instruments.

Electroceutical approach ameliorates intracellular PMP22 aggregation and promotes pro-myelinating pathways in a CMT1A in vitro model.

Charcot-Marie-Tooth disease subtype 1A (CMT1A) is one of the most prevalent demyelinating peripheral neuropathies worldwide, caused by duplication of the peripheral myelin protein 22 (PMP22) gene, which is expressed primarily in Schwann cells (SCs). PMP22 overexpression in SCs leads to intracellular aggregation of the protein, which eventually results in demyelination. Unfortunately, previous biochemical approaches have not resulted in an approved treatment for CMT1A disease, compelling the pursuit for a biophysical approach such as electrical stimulation (ES). However, the effects of ES on CMT1A SCs have remained unexplored. In this study, we established PMP22-overexpressed Schwannoma cells as a CMT1A in vitro model, and investigated the biomolecular changes upon applying ES via a custom-made high-throughput ES platform, screening for the condition that delivers optimal therapeutic effects. While PMP22-overexpressed Schwannoma exhibited intracellular PMP22 aggregation, ES at 20 Hz for 1 h improved this phenomenon, bringing PMP22 distribution closer to healthy condition. ES at this condition also enhanced the expression of the genes encoding myelin basic protein (MBP) and myelin-associated glycoprotein (MAG), which are essential for assembling myelin sheath. Furthermore, ES altered the gene expression for myelination-regulating transcription factors Krox-20, Oct-6, c-Jun and Sox10, inducing pro-myelinating effects in PMP22-overexpressed Schwannoma. While electroceuticals has previously been applied in the peripheral nervous system towards acquired peripheral neuropathies such as pain and nerve injury, this study demonstrates its effectiveness towards ameliorating biomolecular abnormalities in an in vitro model of CMT1A, an inherited peripheral neuropathy. These findings will facilitate the clinical translation of an electroceutical treatment for CMT1A.

The role of PIEZO ion channels in the musculoskeletal system.

PIEZO1 and PIEZO2 are mechanosensitive cation channels that are highly expressed in numerous tissues throughout the body and exhibit diverse, cell-specific functions in multiple organ systems. Within the musculoskeletal system, PIEZO1 functions to maintain muscle and bone mass, sense tendon stretch, and regulate senescence and apoptosis in response to mechanical stimuli within cartilage and the intervertebral disc. PIEZO2 is essential for transducing pain and touch sensations as well as proprioception in the nervous system, which can affect musculoskeletal health. PIEZO1 and PIEZO2 have been shown to act both independently as well as synergistically in different cell types. Conditions that alter PIEZO channel mechanosensitivity, such as inflammation or genetic mutations, can have drastic effects on these functions. For this reason, therapeutic approaches for PIEZO-related disease focus on altering PIEZO1 and/or PIEZO2 activity in a controlled manner, either through inhibition with small molecules, or through dietary control and supplementation to maintain a healthy cell membrane composition. Although many opportunities to better understand PIEZO1 and PIEZO2 remain, the studies summarized in this review highlight how crucial PIEZO channels are to musculoskeletal health and point to promising possible avenues for their modulation as a therapeutic target.

Therapeutic Effect of Three-Dimensional Cultured Adipose-Derived Stem Cell-Conditioned Medium in Renal Ischemia-Reperfusion Injury.

Background and Objectives: We evaluated the effect of adipose-derived stem cell-derived conditioned medium (ADSC-CM) on the renal function of rats with renal ischemia-reperfusion injury (IRI)-induced acute kidney injury. Methods and Results: Forty male Sprague-Dawley rats were randomly divided into four groups: sham, nephrectomy control, IRI control, ADSC-CM. The ADSC-CM was prepared using the three-dimensional spheroid culture system and injected into renal parenchyme. The renal function of the rats was evaluated 28 days before and 1, 2, 3, 4, 7, and 14 days after surgical procedures. The rats were sacrificed 14 days after surgical procedures, and kidney tissues were collected for histological examination. The renal parenchymal injection of ADSC-CM significantly reduced the serum blood urea nitrogen and creatinine levels compared with the IRI control group on days 1, 2, 3, and 4 after IRI. The renal parenchymal injection of ADSC-CM significantly increased the level of creatinine clearance compared with the IRI control group 1 day after IRI. Collagen content was significantly lower in the ADSC-CM group than in the IRI control group in the cortex and medulla. Apoptosis was significantly decreased, and proliferation was significantly increased in the ADSC-CM group compared to the IRI control group in the cortex and medulla. The expressions of anti-oxidative makers were higher in the ADSC-CM group than in the IRI control group in the cortex and medulla. Conclusions: The renal function was effectively rescued through the renal parenchymal injection of ADSC-CM prepared using a three-dimensional spheroid culture system.

Efficacy and Safety of Human Bone Marrow-Derived Mesenchymal Stem Cells according to Injection Route and Dose in a Chronic Kidney Disease Rat Model.

Background and Objectives: We compared the efficacy and safety of human bone marrow-derived mesenchymal stem cells (hBMSC), delivered at different doses and via different injection routes in an animal model of chronic kidney disease. Methods and Results: A total of ninety 12-week-old rats underwent 5/6 nephrectomy and randomized among nine groups: sham, renal artery control (RA-C), tail vein control (TV-C), renal artery low dose (RA-LD) (0.5×106 cells), renal artery moderate dose (RA-MD) (1.0×106 cells), renal artery high dose (RA-HD) (2.0×106 cells), tail vein low dose (TV-LD) (0.5×106 cells), tail vein moderate dose (TV-MD) (1.0×106 cells), and tail vein high dose (TV-HD) (2.0×106 cells). Renal function and mortality of rats were evaluated after hBMSC injection. Serum blood urea nitrogen was significantly lower in the TV-HD group at 2 weeks (p<0.01), 16 weeks (p<0.05), and 24 weeks (p<0.01) than in the TV-C group, as determined by one-way ANOVA. Serum creatinine was significantly lower in the TV-HD group at 24 weeks (p<0.05). At 8 weeks, creatinine clearance was significantly higher in the TV-MD and TV-HD groups (p<0.01, p<0.05) than in the TV-C group. In the safety evaluation, we observed no significant difference among the groups. Conclusions: Our findings confirm the efficacy and safety of high dose (2×106 cells) injection of hBMSC via the tail vein.

Thermogelling hydrogel charge and lower critical solution temperature influence cellular infiltration and tissue integration in an ex vivo cartilage explant model.

Thermogelling hydrogels based on poly(N-isopropyl acrylamide) (p[NiPAAm]) and crosslinked with a peptide-bearing macromer poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) were fabricated to assess the role of hydrogel charge and lower critical solution temperature (LCST) over time in influencing cellular infiltration and tissue integration in an ex vivo cartilage explant model over 21 days. The p(NiPAAm)-based thermogelling polymer was synthesized to possess 0, 5, and 10 mol% dimethyl-γ-butyrolactone acrylate (DBA) to raise the LCST over time as the lactone rings hydrolyzed. Further, three peptides were designed to impart charge into the hydrogels via conjugation to the PdBT crosslinker. The positively, neutrally, and negatively charged peptides K4 (+), zwitterionic K2E2 (0), and E4 (-), respectively, were conjugated to the modular PdBT crosslinker and the hydrogels were evaluated for their thermogelation behavior in vitro before injection into the cartilage explant models. Samples were collected at days 0 and 21, and tissue integration and cellular infiltration were assessed via mechanical pushout testing and histology. Negatively charged hydrogels whose LCST changed over time (10 mol% DBA) were demonstrated to promote the greatest tissue integration when compared to the positive and neutral gels of the same thermogelling polymer formulation due to increased transport and diffusion across the hydrogel-tissue interface. Indeed, the negatively charged thermogelling polymer groups containing 5 and 10 mol% DBA demonstrated cellular infiltration and cartilage-like matrix deposition via histology. This study demonstrates the important role that material physicochemical properties play in dictating cell and tissue behavior and can inform future cartilage tissue engineering strategies.

Engineering Hyaluronic Acid for the Development of New Treatment Strategies for Osteoarthritis.

Osteoarthritis (OA) is a degenerative joint disease that is characterized by inflammation of the joints, degradation of cartilage, and the remodeling of other joint tissues. Due to the absence of disease-modifying drugs for OA, current clinical treatment options are often only effective at slowing down disease progression and focus mainly on pain management. The field of tissue engineering has therefore been focusing on developing strategies that could be used not only to alleviate symptoms of OA but also to regenerate the damaged tissue. Hyaluronic acid (HA), an integral component of both the synovial fluid and articular cartilage, has gained widespread usage in developing hydrogels that deliver cells and biomolecules to the OA joint thanks to its biocompatibility and ability to support cell growth and the chondrogenic differentiation of encapsulated stem cells, providing binding sites for growth factors. Tissue-engineering strategies have further attempted to improve the role of HA as an OA therapeutic by developing diverse modified HA delivery platforms for enhanced joint retention and controlled drug release. This review summarizes recent advances in developing HA-based hydrogels for OA treatment and provides additional insights into how HA-based therapeutics could be further improved to maximize their potential as a viable treatment option for OA.

Implantable Electroceutical Approach Improves Myelination by Restoring Membrane Integrity in a Mouse Model of Peripheral Demyelinating Neuropathy.

Although many efforts are undertaken to treat peripheral demyelinating neuropathies based on biochemical interventions, unfortunately, there is no approved treatment yet. Furthermore, previous studies have not shown improvement of the myelin membrane at the biomolecular level. Here, an electroceutical treatment is introduced as a biophysical intervention to treat Charcot-Marie-Tooth (CMT) disease-the most prevalent peripheral demyelinating neuropathy worldwide-using a mouse model. The specific electrical stimulation (ES) condition (50 mV mm-1 , 20 Hz, 1 h) for optimal myelination is found via an in vitro ES screening system, and its promyelinating effect is validated with ex vivo dorsal root ganglion model. Biomolecular investigation via time-of-flight secondary ion mass spectrometry shows that ES ameliorates distribution abnormalities of peripheral myelin protein 22 and cholesterol in the myelin membrane, revealing the restoration of myelin membrane integrity. ES intervention in vivo via flexible implantable electrodes shows not only gradual rehabilitation of mouse behavioral phenotypes (balance and endurance), but also restored myelin thickness, compactness, and membrane integrity. This study demonstrates, for the first time, that an electroceutical approach with the optimal ES condition has the potential to treat CMT disease and restore impaired myelin membrane integrity, shifting the paradigm toward practical interventions for peripheral demyelinating neuropathies.

A fully automated primary neuron purification system using continuous centrifugal microfluidics.

Progress in neurological research has experienced bottlenecks owing to the limited availability of purified primary neurons. Since neuronal cells are non-proliferative, it is necessary to obtain purified neurons from animal models or human patients for experimental work. However, currently available methods for purifying primary neurons are time-consuming (taking approximately 1 week), and suffer from insufficient viability and purity. Here, we report a method for rapid enrichment of neurons from the mouse embryonic dorsal root ganglion (DRG), using a fully-automated continuous centrifugal microfluidics (CCM) based neuron purification disc (NPD). Non-neuronal cells were removed via negative depletion by combining density gradient centrifugation and immunomagnetic separation. The CCM-NPD platform enables effective isolation of intact neurons within 13 min, which is approximately 800 times faster than the conventional chemical purification method. Furthermore, the neurons purified using the CCM-NPD platform showed better neurite growth, along with higher viability (93.5%) and purity (97.0%) after 1 week of culture, compared to the chemical purification method. Therefore, the proposed automated and rapid system yields purified DRG neurons with high viability and purity, while avoiding the use of harsh chemicals. We believe this system will significantly mitigate the shortage of purified primary neurons and advance neurological research.

Advantages of Adult Mouse Dorsal Root Ganglia Explant Culture in Investigating Myelination in an Inherited Neuropathic Mice Model.

A co-culture of neurons and Schwann cells has frequently been used to investigate myelin sheath formation. However, this approach is restricted to myelin-related diseases of the peripheral nervous system. This study introduces and compares an ex vivo model of adult-mouse-derived dorsal root ganglia (DRG) explant, with an in vitro co-culture of dissociated neurons from mouse embryo DRG and Schwann cells from a mouse sciatic nerve. The 2D co-culture has disadvantages of different mouse isolation for neurons and Schwann cells, animal number, culture duration, and the identification of disease model. However, 3D DRG explant neurons and myelination cells in Matrigel-coated culture are obtained from the same mouse, the culture period is shorter than that of 2D co-culture, and fewer animals are needed. In addition, it has simpler and shorter experimental steps than 2D co-culture. This culture system may prove advantageous in studies of biological functions and pathophysiological mechanisms of disease models, since it can reflect disease characteristics as traditional co-culture does. Therefore, it is suggested that a DRG explant culture is a scientifically, ethically, and economically more practical option than a co-culture system for studying myelin dynamics, myelin sheath formation, and demyelinating disease.

Comparison of Stromal Vascular Fraction and Adipose-Derived Stem Cells for Protection of Renal Function in a Rodent Model of Ischemic Acute Kidney Injury.

Aims: Few studies have compared the use of different cell types derived from adipose tissue or the optimal route for efficient and safe cell delivery in ischemic acute kidney injury (AKI). We compared the abilities of stromal vascular fraction (SVF) and adipose-derived stem cells (ADSC), injected via three different routes, to protect renal function in a rodent model of ischemic AKI. Methods: Ninety male Sprague-Dawley rats were randomly divided into 9 groups: sham, nephrectomy control, AKI control, transaortic renal arterial SVF injection, renal parenchymal SVF injection, tail venous SVF injection, transaortic renal arterial ADSC injection, renal parenchymal ADSC injection, and tail venous ADSC injection groups. Their renal function was assessed 4 days before and 1, 2, 3, 4, 7, and 14 days after surgical procedures to induce ischemic AKI. The histomorphometric studies were performed 14 days after surgical procedures. Results: Renal parenchymal injection of SVF notably reduced the level of serum blood urea nitrogen and creatinine elevation compared to the AKI control group. Renal parenchymal injection of SVF notably reduced the level of creatinine clearance decrease. In addition, collagen content was lower in the renal parenchymal SVF injection group, and fibrosis was reduced. Apoptosis was reduced in the renal parenchymal SVF injection group, and proliferation was increased. The expression levels of antioxidative markers such as glutathione reductase and peroxidase were higher in the renal parenchymal SVF injection group. Conclusions: Our findings suggest that renal function is protected from ischemic AKI through renal parenchymal injection of SVF, which has enhanced antifibrotic, antiapoptotic, and antioxidative effects.

A dual-gelling poly(N-isopropylacrylamide)-based ink and thermoreversible poloxamer support bath for high-resolution bioprinting.

Extrusion bioprinting is a popular method for fabricating tissue engineering scaffolds because of its potential to rapidly produce complex, bioactive or cell-laden scaffolds. However, due to the relatively high viscosity required to maintain shape fidelity during printing, many extrusion-based inks lack the ability to achieve precise structures at scales lower than hundreds of micrometers. In this work, we present a novel poly(N-isopropylacrylamide) (PNIPAAm)-based ink and poloxamer support bath system that produces precise, multi-layered structures on the tens of micrometers scale. The support bath maintains the structure of the ink in a hydrated, heated environment ideal for cell culture, while the ink undergoes rapid thermogelation followed by a spontaneous covalent crosslinking reaction. Through the combination of the PNIPAAm-based ink and poloxamer bath, this system was able to produce hydrogel scaffolds with uniform fibers possessing diameters tunable from 80 to 200 µm. A framework of relationships between several important printing factors involved in maintaining support and thermogelation was also elucidated. As a whole, this work demonstrates the ability to produce precise, acellular and cell-laden PNIPAAm-based scaffolds at high-resolution and contributes to the growing body of research surrounding the printability of extrusion-based bioinks with support baths.

Solo-surgeon pure laparoscopic donor nephrectomy using passive camera holder: IDEAL stage 2a study.

BACKGROUND: Solo-surgery can be defined as a practice of a surgeon operating alone using a camera holder, without other surgical members except for a scrub nurse. This study was designed to evaluate the feasibility and safety of solo-surgeon pure laparoscopic donor nephrectomy. METHODS: The study protocol was approved by the Institutional Review Board of Asan Medical Center, Seoul, Korea. The brief study protocol was registered on the Clinical Research Information Service site of the Korea Centers for Disease Control and Prevention. Candidates fulfilling all inclusion and exclusion criteria were enrolled in the clinical trial and underwent solo-surgeon pure laparoscopic donor nephrectomy. The feasibility was assessed by the proportion of subjects who could undergo solo-surgeon pure laparoscopic donor nephrectomy without difficulty. The perioperative complications were identified to assess the safety of solo-surgeon pure laparoscopic donor nephrectomy. RESULTS: Of the 47 potential candidates from November 2018 to August 2019, 40 were enrolled in the clinical trial and seven excluded due to declining participation. The feasibility of solo-surgeon pure laparoscopic donor nephrectomy was 100%, without an occasion of any difficulty requiring conversion to the human assisted pure laparoscopic donor nephrectomy. Fourteen intraoperative complications occurred in 10 patients. The most common intraoperative complication was spleen injury. Two of three cases classified as the Satava classification grade II were due to the incomplete stapling of endoscopic stapler. Seventy-eight postoperative complications occurred in 34 patients. The most common postoperative complication was nausea/vomiting and followed by aspartate aminotransferase/alanine aminotransferase elevation. Most postoperative complication was independent of the solo-surgery itself. CONCLUSIONS: Solo-surgeon pure laparoscopic donor nephrectomy using passive camera holder is technically feasible. In terms of safety, it is necessary to adjust the scope of surgery performed alone. Trial Registration CRIS, KCT0003458. Registered 30/01/2019, Retrospectively registered, https://cris.nih.go.kr/cris/search/detailSearch.do/15868 .

Therapeutic Effect of Human Mesenchymal Stem Cell-Conditioned Medium on Erectile Dysfunction.

PURPOSE: Owing to the safety and cost effectiveness of conditioned medium (CM), its therapeutic effects have attracted significant attention from many researchers. To date, numerous studies have been conducted on CM; however, little has been done with regard to erectile dysfunction (ED). In this research, the potential of human mesenchymal stem cell-derived CM (MSC-CM) for the treatment of ED was investigated. MATERIALS AND METHODS: A high concentration of MSC-CM was prepared through 3D spheroid culturing with bone marrow-derived MSCs and cut-off filtering. The composition of CM was analyzed using biochemical assays, and the effect of the preparation process on the quality of CM was investigated. The therapeutic effects of MSC-CM were evaluated through animal studies using a cavernous nerve (CN) injury rat model. RESULTS: 3D spheroid culturing afforded a 278-fold increase in the total protein content of CM, as compared to that from 2D cultures; the protein concentration increased by 19 times on increasing the centrifugation time for cut-off filtering. Biochemical assays indicated that the CM contains various types of angiogenic, neurotrophic, and anti-inflammatory factors. Histological assay results showed that MSC-CM has angio- and neuro-trophic effect in a CN injury rat model in vivo, and these therapeutic effects appear in a dose-dependent manner. CONCLUSIONS: The experimental results confirmed the therapeutic effect of MSC-CM in healing damaged cavernosal tissue and restoring erectile function. These results successfully demonstrated that MSC-CM has significant potential for the treatment of ED.

An electroceutical approach enhances myelination via upregulation of lipid biosynthesis in the dorsal root ganglion.

As the myelin sheath is crucial for neuronal saltatory conduction, loss of myelin in the peripheral nervous system (PNS) leads to demyelinating neuropathies causing muscular atrophy, numbness, foot deformities and paralysis. Unfortunately, few interventions are available for such neuropathies, because previous pharmaceuticals have shown severe side effects and failed in clinical trials. Therefore, exploring new strategies to enhance PNS myelination is critical to provide solution for such intractable diseases. This study aimed to investigate the effectiveness of electrical stimulation (ES) to enhance myelination in the mouse dorsal root ganglion (DRG)-anex vivomodel of the PNS. Mouse embryonic DRGs were extracted at E13 and seeded onto Matrigel-coated surfaces. After sufficient growth and differentiation, screening was carried out by applying ES in the 1-100 Hz range at the beginning of the myelination process. DRG myelination was evaluated via immunostaining at the intermediate (19 daysin vitro(DIV)) and mature (30 DIV) stages. Further biochemical analyses were carried out by utilizing ribonucleic acid sequencing, quantitative polymerase chain reaction and biochemical assays at both intermediate and mature myelination stages. Imaging of DRG myelin lipids was carried out via time-of-flight secondary ion mass spectrometry (ToF-SIMS). With screening ES conditions, optimal condition was identified at 20 Hz, which enhanced the percentage of myelinated neurons and average myelin length not only at intermediate (129% and 61%) but also at mature (72% and 17%) myelination stages. Further biochemical analyses elucidated that ES promoted lipid biosynthesis in the DRG. ToF-SIMS imaging showed higher abundance of the structural lipids, cholesterol and sphingomyelin, in the myelin membrane. Therefore, promotion of lipid biosynthesis and higher abundance of myelin lipids led to ES-mediated myelination enhancement. Given that myelin lipid deficiency is culpable for most demyelinating PNS neuropathies, the results might pave a new way to treat such diseases via electroceuticals.

Evaluating the physicochemical effects of conjugating peptides into thermogelling hydrogels for regenerative biomaterials applications.

Thermogelling hydrogels, such as poly(N-isopropylacrylamide) [P(NiPAAm)], provide tunable constructs leveraged in many regenerative biomaterial applications. Recently, our lab developed the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol), which crosslinks P(NiPAAm-co-glycidyl methacrylate) via thiol-epoxy reaction and can be functionalized with azide-terminated peptides via alkyne-azide click chemistry. This study's aim was to evaluate the impact of peptides on the physicochemical properties of the hydrogels. The physicochemical properties of the hydrogels including the lower critical solution temperature, crosslinking times, swelling, degradation, peptide release and cytocompatibility were evaluated. The gels bearing peptides increased equilibrium swelling indicating hydrophilicity of the hydrogel components. Comparable sol fractions were found for all groups, indicating that inclusion of peptides does not impact crosslinking. Moreover, the inclusion of a matrix metalloproteinase-sensitive peptide allowed elucidation of whether release of peptides from the network was driven by hydrolysis or enzymatic cleavage. The hydrophilicity of the network determined by the swelling behavior was demonstrated to be the most important factor in dictating hydrogel behavior over time. This study demonstrates the importance of characterizing the impact of additives on the physicochemical properties of hydrogels. These characteristics are key in determining design considerations for future in vitro and in vivo studies for tissue regeneration.

Development of a modular, biocompatible thiolated gelatin microparticle platform for drug delivery and tissue engineering applications.

The field of biomaterials has advanced significantly in the past decade. With the growing need for high-throughput manufacturing and screening, the need for modular materials that enable streamlined fabrication and analysis of tissue engineering and drug delivery schema has emerged. Microparticles are a powerful platform that have demonstrated promise in enabling these technologies without the need to modify a bulk scaffold. This building block paradigm of using microparticles within larger scaffolds to control cell ratios, growth factors and drug release holds promise. Gelatin microparticles (GMPs) are a well-established platform for cell, drug and growth factor delivery. One of the challenges in using GMPs though is the limited ability to modify the gelatin post-fabrication. In the present work, we hypothesized that by thiolating gelatin before microparticle formation, a versatile platform would be created that preserves the cytocompatibility of gelatin, while enabling post-fabrication modification. The thiols were not found to significantly impact the physicochemical properties of the microparticles. Moreover, the thiolated GMPs were demonstrated to be a biocompatible and robust platform for mesenchymal stem cell attachment. Additionally, the thiolated particles were able to be covalently modified with a maleimide-bearing fluorescent dye and a peptide, demonstrating their promise as a modular platform for tissue engineering and drug delivery applications.

Evaluation of tissue integration of injectable, cell-laden hydrogels of cocultures of mesenchymal stem cells and articular chondrocytes with an ex vivo cartilage explant model.

This study investigated the chondrogenic activity of encapsulated mesenchymal stem cells (MSCs) and articular chondrocytes (ACs) and its impact on the mechanical properties of injectable poly(N-isopropylacrylamide)-based dual-network hydrogels loaded with poly( l -lysine) (PLL). To this effect, an ex vivo study model was employed to assess the behavior of the injected hydrogels-specifically, their surface stiffness and integration strength with the surrounding cartilage. The highest chondrogenic activity was observed from AC-encapsulated hydrogels, while the effect of PLL on MSC chondrogenesis was not apparent from biochemical analyses. Mechanical testing showed that there were no significant differences in either surface stiffness or integration strength among the different study groups. Altogether, the results suggest that the ex vivo model can allow further understanding of the relationship between biochemical changes within the hydrogel and their impact on the hydrogel's mechanical properties.

Bilayered, peptide-biofunctionalized hydrogels for in vivo osteochondral tissue repair.

Osteochondral defects present a unique clinical challenge due to their combination of phenotypically distinct cartilage and bone, which require specific, stratified biochemical cues for tissue regeneration. Furthermore, the articular cartilage exhibits significantly worse regeneration than bone due to its largely acellular and avascular nature, prompting significant demand for regenerative therapies. To address these clinical challenges, we have developed a bilayered, modular hydrogel system that enables the click functionalization of cartilage- and bone-specific biochemical cues to each layer. In this system, the crosslinker poly(glycolic acid)-poly(ethylene glycol)-poly(glycolic acid)-di(but-2-yne-1,4-dithiol) (PdBT) was click conjugated with either a cartilage- or bone-specific peptide sequence of interest, and then mixed with a suspension of thermoresponsive polymer and mesenchymal stem cells (MSCs) to generate tissue-specific, cell-encapsulated hydrogel layers targeting the cartilage or bone. We implanted bilayered hydrogels in rabbit femoral condyle defects and investigated the effects of tissue-specific peptide presentation and cell encapsulation on osteochondral tissue repair. After 12 weeks implantation, hydrogels with a chondrogenic peptide sequence produced higher histological measures of overall defect filling, cartilage surface regularity, glycosaminoglycan (GAG)/cell content of neocartilage and adjacent cartilage, and bone filling and bonding compared to non-chondrogenic hydrogels. Furthermore, MSC encapsulation promoted greater histological measures of overall defect filling, cartilage thickness, GAG/cell content of neocartilage, and bone filling. Our results establish the utility of this click functionalized hydrogel system for in vivo repair of the osteochondral unit. STATEMENT OF SIGNIFICANCE: Osteochondral repair requires mimicry of both cartilage- and bone-specific biochemical cues, which are highly distinct. While traditional constructs for osteochondral repair have mimicked gross compositional differences between the cartilage and bone in mineral content, mechanical properties, proteins, or cell types, few constructs have recapitulated the specific biochemical cues responsible for the differential development of cartilage and bone. In this study, click biofunctionalized, bilayered hydrogels produced stratified presentation of developmentally inspired peptide sequences for chondrogenesis and osteogenesis. This work represents, to the authors' knowledge, the first application of bioconjugation chemistry for the simultaneous repair of bone and cartilage tissue. The conjugation of tissue-specific peptide sequences successfully promoted development of both cartilage and bone tissues in vivo.