Biomimetic Proteoglycans Strengthen the Pericellular Matrix of Normal and Osteoarthritic Human Cartilage.

In osteoarthritis (OA), degradation of cartilage pericellular matrix (PCM), the proteoglycan-rich immediate cell microniche, is a leading event of disease initiation. This study demonstrated that biomimetic proteoglycans (BPGs) can diffuse into human cartilage from both normal and osteoarthritic donors and are preferentially localized within the PCM. Applying immunofluorescence (IF)-guided AFM nanomechanical mapping, we show that this localization of BPGs increases the PCM micromodulus of both normal and OA specimens. These results illustrate the capability of BPGs to integrate with degenerative tissues and support the translational potential of BPGs for treating human OA and other diseases associated with proteoglycan degradation.

Biomimetic proteoglycans as a tool to engineer the structure and mechanics of porcine bioprosthetic heart valves.

The utility of bioprosthetic heart valves (BHVs) is limited to certain patient populations because of their poor durability compared to mechanical prosthetic valves. Histological analysis of failed porcine BHVs suggests that degeneration of the tissue extracellular matrix (ECM), specifically the loss of proteoglycans and their glycosaminoglycans (GAGs), may lead to impaired mechanical performance, resulting in nucleation and propagation of tears and ultimately failure of the prosthetic. Several strategies have been proposed to address this deterioration, including novel chemical fixatives to stabilize ECM constituents and incorporation of small molecule inhibitors of catabolic enzymes implicated in the degeneration of the BHV ECM. Here, biomimetic proteoglycans (BPGs) were introduced into porcine aortic valves ex vivo and were shown to distribute throughout the valve leaflets. Incorporation of BPGs into the heart valve leaflet increased tissue overall GAG content. The presence of BPGs also significantly increased the micromodulus of the spongiosa layer within the BHV without compromising the chemical fixation process used to sterilize and strengthen the tissue prior to implantation. These findings suggest that a targeted approach for molecularly engineering valve leaflet ECM through the use of BPGs may be a viable way to improve the mechanical behavior and potential durability of BHVs.

Targeting cell-matrix interface mechanobiology by integrating AFM with fluorescence microscopy.

Mechanosensing at the interface of a cell and its surrounding microenvironment is an essential driving force of physiological processes. Understanding molecular activities at the cell-matrix interface has the potential to provide novel targets for improving tissue regeneration and early disease intervention. In the past few decades, the advancement of atomic force microscopy (AFM) has offered a unique platform for probing mechanobiology at this crucial microdomain. In this review, we describe key advances under this topic through the use of an integrated system of AFM (as a biomechanical testing tool) with complementary immunofluorescence (IF) imaging (as an in situ navigation system). We first describe the body of work investigating the micromechanics of the pericellular matrix (PCM), the immediate cell micro-niche, in healthy, diseased, and genetically modified tissues, with a focus on articular cartilage. We then summarize the key findings in understanding cellular biomechanics and mechanotransduction, in which, molecular mechanisms governing transmembrane ion channel-mediated mechanosensing, cytoskeleton remodeling, and nucleus remodeling have been studied in various cell and tissue types. Lastly, we provide an overview of major technical advances that have enabled more in-depth studies of mechanobiology, including the integration of AFM with a side-view microscope, multiple optomicroscopy, a fluorescence recovery after photobleaching (FRAP) module, and a tensile stretching device. The innovations described here have contributed greatly to advancing the fundamental knowledge of extracellular matrix biomechanics and cell mechanobiology for improved understanding, detection, and intervention of various diseases.

Insight on the Role of Poly(acrylic acid) for Directing Calcium Phosphate Mineralization of Synthetic Polymer Bone Scaffolds.

Bone is a complex tissue with robust mechanical and biological properties originating from its nanoscale composite structure. Although much research has been conducted on designing bioinspired artificial bone, the role of biological macromolecules such as noncollagenous proteins (NCPs) in influencing the formation of biominerals is not fully understood. In this work, we have designed nanofiber shish-kebab (NFSK) structures that can template mineral location by recruiting calcium cations from an ion-rich mineralization solution. Poly(acrylic acid) (PAA) is used as the NCP analogue to understand the role of polyelectrolytes in scaffold mineralization. We demonstrate that the addition of PAA in the mineralization solution suppresses the development of extrafibrillar minerals as well as slows down the accumulation and development of mineral phases within NFSKs. We probe the mechanism behind this effect by monitoring the free calcium ion concentration, investigating the PAA molecular weight effect, and conducting mineralization in membrane-partitioned solutions. Our results suggest the 2-fold effect of PAA as a solution stabilizer and physical barrier on the NFSK surface. This work could shed light on the understanding of the NCP effect in biomineralization.

Molecular Engineering of Pericellular Microniche via Biomimetic Proteoglycans Modulates Cell Mechanobiology.

Molecular engineering of biological tissues using synthetic mimics of native matrix molecules can modulate the mechanical properties of the cellular microenvironment through physical interactions with existing matrix molecules, and in turn, mediate the corresponding cell mechanobiology. In articular cartilage, the pericellular matrix (PCM) is the immediate microniche that regulates cell fate, signaling, and metabolism. The negatively charged osmo-environment, as endowed by PCM proteoglycans, is a key biophysical cue for cell mechanosensing. This study demonstrated that biomimetic proteoglycans (BPGs), which mimic the ultrastructure and polyanionic nature of native proteoglycans, can be used to molecularly engineer PCM micromechanics and cell mechanotransduction in cartilage. Upon infiltration into bovine cartilage explant, we showed that localization of BPGs in the PCM leads to increased PCM micromodulus and enhanced chondrocyte intracellular calcium signaling. Applying molecular force spectroscopy, we revealed that BPGs integrate with native PCM through augmenting the molecular adhesion of aggrecan, the major PCM proteoglycan, at the nanoscale. These interactions are enabled by the biomimetic "bottle-brush" ultrastructure of BPGs and facilitate the integration of BPGs within the PCM. Thus, this class of biomimetic molecules can be used for modulating molecular interactions of pericellular proteoglycans and harnessing cell mechanosensing. Because the PCM is a prevalent feature of various cell types, BPGs hold promising potential for improving regeneration and disease modification for not only cartilage-related healthcare but many other tissues and diseases.

Painful temporomandibular joint overloading induces structural remodeling in the pericellular matrix of that joint's chondrocytes.

Mechanical stress to the temporomandibular joint (TMJ) is an important factor in cartilage degeneration, with both clinical and preclinical studies suggesting that repeated TMJ overloading could contribute to pain, inflammation, and/or structural damage in the joint. However, the relationship between pain severity and early signs of cartilage matrix microstructural dysregulation is not understood, limiting the advancement of diagnoses and treatments for temporomandibular joint-osteoarthritis (TMJ-OA). Changes in the pericellular matrix (PCM) surrounding chondrocytes may be early indicators of OA. A rat model of TMJ pain induced by repeated jaw loading (1 h/day for 7 days) was used to compare the extent of PCM modulation for different loading magnitudes with distinct pain profiles (3.5N-persistent pain, 2N-resolving pain, or unloaded controls-no pain) and macrostructural changes previously indicated by Mankin scoring. Expression of PCM structural molecules, collagen VI and aggrecan NITEGE neo-epitope, were evaluated at Day 15 by immunohistochemistry within TMJ fibrocartilage and compared between pain conditions. Pericellular collagen VI levels increased at Day 15 in both the 2N (p = 0.003) and 3.5N (p = 0.042) conditions compared to unloaded controls. PCM width expanded to a similar extent for both loading conditions at Day 15 (2N, p < 0.001; 3.5N, p = 0.002). Neo-epitope expression increased in the 3.5N group over levels in the 2N group (p = 0.041), indicating pericellular changes that were not identified in the same groups by Mankin scoring of the pericellular region. Although remodeling occurs in both pain conditions, the presence of pericellular catabolic neo-epitopes may be involved in the macrostructural changes and behavioral sensitivity observed in persistent TMJ pain.

MC3T3 E1 cell response to mineralized nanofiber shish kebab structures.

Block copolymers (BCPs) are of growing interest because of their extensive utility in tissue engineering, particularly in biomimetic approaches where multifunctionality is critical. We synthesized polycaprolactone-polyacrylic acid (PCL-b-PAA) BCP and crystallized it onto PCL nanofibers, making BCP nanofiber shish kebab (BCP NFSK) structures. When mineralized in 2× simulated body fluid, BCP NFSK mimic the structure of mineralized collagen fibrils. We hypothesized that the addition of a calcium phosphate layer of graded roughness on the nano-structure of the nanofiber shish kebabs would enhance preosteoblast alkaline phosphatase (ALP) activity, which has been shown to be a critical component in bone matrix formation. The objectives in the study were to investigate the effect of mineralization on cell proliferation and ALP activity, and to also investigate the effect of BCP NFSK periodicity, a structural feature describing the distance between PCL-b-PAA crystals on the nanofiber core, on cell proliferation, and ALP activity. ALP activity of cells cultured on the mineralized BCP NFSK template was significantly higher than the nonmineralized BCP NFSK templates. Interestingly, no statistical difference was observed in ALP activity when the periodic varied, indicating that surface chemistry seemed to play a larger role than the surface roughness.

A Cross University-Led COVID-19 Rapid-Response Effort: Design, Build, and Distribute Drexel AJFlex Face Shields.

The purpose of this article is to demonstrate how a new cross-community leadership team came together, collaborated, coordinated across academic units with external community partners, and executed a joint mission to address the unmet clinical need for medical face shields during these unprecedented times. Key aspects of this success include the ability to forge and leverage new opportunities, overcome challenges, adapt to changing constraints, and serve the significant need across the Philadelphia region and healthcare systems. We teamed to design-build durable face shields (AJFlex Shields). This was accomplished by high-volume manufacturing via injection molding and by 3-D printing the key headband component that supports the protective shield. Partnering with industry collaborators and civic-minded community allies proved to be essential to bolster production and deliver approximately 33,000 face shields to more than 100 organizations in the region. Our interdisciplinary team of engineers, clinicians, product designers, manufacturers, distributors, and dedicated volunteers is committed to continuing the design-build effort and providing Drexel AJFlex Shields to our communities.

New materials for hip and knee joint replacement: What's hip and what's in kneed?

Over the last three decades there have been significant advancements in the knee and hip replacement technology that has been driven by an issue in the past concerning adverse local tissue reactions, aseptic and septic loosening. The implants and the materials we utilize have improved over the last two decades and in knee and hip replacement there has been a decrease in the failures attributed to wear and osteolysis. Despite these advancements there are still issues with patient satisfaction and early revisions due to septic and aseptic loosening in knee replacement patients. This article reviews the state of current implant material technology in hip and knee replacement surgery, discusses some of the unmet needs we have in biomaterials, and reviews some of the current biomaterials and technology that may be able to solve the most common issues in the knee and hip replacement surgery.

Does annealing improve the interlayer adhesion and structural integrity of FFF 3D printed PEEK lumbar spinal cages?

Polyaryletheretherketone (PEEK) has been commonly used for interbody fusion devices because of its biocompatibility, radiolucency, durability, and strength. Although the technology of PEEK Additive Manufacturing (AM) is rapidly developing, post-processing techniques of 3D printed PEEK remain poorly understood. AM of PEEK has been challenging because of its high melt temperature (over 340 °C) and requires specialized equipment which was not commercially available until recently. A lumbar fusion cage design, used in ASTM interlaboratory studies, was 3D printed with a medical grade PEEK filament via Fused Filament Fabrication (FFF) under two different print speeds. PEEK cages were then annealed above the PEEK's glass transition temperature, at 200 °C or 300 °C. AM cages were CT scanned to determine the porosity before and after annealing. Mechanical tests were conducted on cages according to ASTM F2077 (ASTM F2077, 2014). SEM images helped to evaluate the cages' surface morphology before and after heat treatment. It was observed that annealing did not produce markedly better mechanical properties at either temperature, however, it had an effect on the cages' mechanical properties at lower printing speed under all loading conditions. Although the structure of the pores changed after annealing, annealing conditions examined here as a post-processing method were not able to decrease the undesired porosity formed during the 3D printing process or change the failure mechanism, which is due interlayer debonding.

Type III collagen is a key regulator of the collagen fibrillar structure and biomechanics of articular cartilage and meniscus.

Despite the fact that type III collagen is the second most abundant collagen type in the body, its contribution to the physiologic maintenance and repair of skeletal tissues remains poorly understood. This study queried the role of type III collagen in the structure and biomechanical functions of two structurally distinctive tissues in the knee joint, type II collagen-rich articular cartilage and type I collagen-dominated meniscus. Integrating outcomes from atomic force microscopy-based nanomechanical tests, collagen fibril nanostructural analysis, collagen cross-link analysis and histology, we elucidated the impact of type III collagen haplodeficiency on the morphology, nanostructure and biomechanical properties of articular cartilage and meniscus in Col3a1+/- mice. Reduction of type III collagen leads to increased heterogeneity and mean thickness of collagen fibril diameter, as well as reduced modulus in both tissues, and these effects became more pronounced with skeletal maturation. These data suggest a crucial role of type III collagen in mediating fibril assembly and biomechanical functions of both articular cartilage and meniscus during post-natal growth. In articular cartilage, type III collagen has a marked contribution to the micromechanics of the pericellular matrix, indicating a potential role in mediating the early stage of type II collagen fibrillogenesis and chondrocyte mechanotransduction. In both tissues, reduction of type III collagen leads to decrease in tissue modulus despite the increase in collagen cross-linking. This suggests that the disruption of matrix structure due to type III collagen deficiency outweighs the stiffening of collagen fibrils by increased cross-linking, leading to a net negative impact on tissue modulus. Collectively, this study is the first to highlight the crucial structural role of type III collagen in both articular cartilage and meniscus extracellular matrices. We expect these results to expand our understanding of type III collagen across various tissue types, and to uncover critical molecular components of the microniche for regenerative strategies targeting articular cartilage and meniscus repair.

Correction to: Translating Mechanobiology to the Clinic: A Panel Discussion from the 2018 CMBE Conference.

[This corrects the article DOI: 10.1007/s12195-018-0556-5.].

Modulating physical properties of porcine urethra with injection of novel biomimetic proteoglycans ex vivo.

Urinary incontinence is a significant challenge for women who are affected by it. We propose augmenting the tissue structure to restore normal biomechanics by molecularly engineering the tissue using a novel family of biomimetic proteoglycans (BPGs). This work examines the ability of BPGs to modulate the mechanical and physical properties of porcine urethras ex vivo to determine the feasibility of BPGs to be implemented as molecular treatment for stress urinary incontinence (SUI). We investigated compliance by performing a unique radial expansion testing method using urethras from six- to nine-month-old pigs. The urethras were injected with 0.5 ml BPG solution at three sites every approximately 120° (conc.: 25 mg ml-1, 50 mg ml-1 and 75 mg ml-1 in 1× phosphate-buffered saline (PBS); n = 4 per group) and compared them with PBS-injected controls. Young's modulus was calculated by treating the urethra as a thin-walled pressure vessel. A water uptake study was performed by soaking 10 mm urethra biopsy samples that were injected with 0.1 ml BPG solution (conc.: 50 mg ml-1, 100 mg ml-1 and 200 mg ml-1 in 1× PBS; n = 6 per group) in 5 ml PBS for 24 h. Although there was no significant difference in Young's modulus data, there were differences between groups as can be seen in the raw radial expansion testing data. Results showed that BPGs have the potential to increase hydration in samples, and that there was a significant difference in water uptake between BPG-injected samples and the controls (100 mg ml-1 samples versus PBS samples, p < 0.05). This work shows that BPGs have the potential to be implemented as a molecular treatment for SUI, by restoring the diminished proteoglycan content and subsequently increasing hydration and improving the compliance of urethral tissue.

Biomimetic proteoglycans diffuse throughout articular cartilage and localize within the pericellular matrix.

Biomimetic proteoglycan (BPG) diffusion into articular cartilage has the potential to restore the lost proteoglycan content in osteoarthritic cartilage given these molecules mimic the structure and properties of natural proteoglycans. We examined the diffusion characteristics of BPGs through cartilage with the use of a custom-made in vitro cartilage diffusion model in both normal bovine and human osteoarthritic cartilage explants. BPGs were introduced into the cartilage through essentially one-dimensional diffusion using osteochondral plugs. The molecular diffusion was shown to be size and concentration dependent. Diffusion profiles were found over different diffusion time intervals and the profiles were fit to a nonlinear Fickian diffusion model. Steady state 011012-7diffusion coefficients for BPGs were found to be 4.01 and 3.53 µm2 /s for 180 and 1600 kDa BPGs, respectfully, and these values are similar to other large molecule diffusion in cartilage. In both bovine and osteoarthritic human cartilage, BPGs were found localized around the chondrocytes. BPG localization was examined by labeling collagen type VI and soaking 5 µm thick sections of cartilage with BPG solutions demonstrating that the BPGs diffused into the cartilage and preferentially localized alongside collagen type VI in the pericellular matrix.

The regulatory effects of proteoglycans on collagen fibrillogenesis and morphology investigated using biomimetic proteoglycans.

Collagen is one of the leading components in extracellular matrix (ECM), providing durability, structural integrity, and functionality for many tissues. Regulation of collagen fibrillogenesis and degradation is important for treating several diseases from orthopedic injuries to genetic deficiencies. In vivo, this process is generally regulated by proteoglycans (PGs), a family of molecules that contain both protein and glycosaminoglycan components. Recently, novel, biocompatible, semi-synthetic biomimetic proteoglycans (BPGs) were developed, which consist of an enzymatically resistant synthetic polymer core and natural chondroitin sulfate (CS) bristles. It was demonstrated that BPGs affect type I collagen fibrillogenesis in vitro, as reflected by their impact delaying the kinetic formation of gels similar to native PGs. To elucidate the interaction and the effect of BPGs on the quality of collagen fibrils, a histological technique, electron tomography, was adapted and utilized to image nano-scale structures in 2D and 3D within the tissue model. BPGs were found to aid in lateral growth and enhance fibril banding periodicity resulting in structures resembling those in native tissue. BPGs attached to collagen despite the lack of a protein core. This interaction was mediated by the CS bristle regions of the BPGs, implying that CS itself is sufficient for PG-type I tropocollagen interactions, in the absence of the protein core, with the overall nanoarchitecture of the molecule serving to affect ECM kinetics. Synthetic mimics are a tool to study non-proteinaceous PG interactions in collagen assembly and warrant exploration as a viable pathway to augmenting molecular repair in collagen type I-rich tissues.

In vivo molecular engineering of the urethra for treatment of stress incontinence using novel biomimetic proteoglycans.

Stress urinary incontinence (SUI), a serious condition which affects ~56% of postmenopausal women, is the involuntary leakage of urine through urethra during physical activity that causes an increase in abdominal pressure. SUI is associated with a decrease in compliance and volume of urethral tissue, likely due to a reduced proteoglycan: collagen ratio in the extracellular matrix and collagen disorganization. Here, we investigated the use of biomimetic proteoglycans (BPGs) to molecularly engineer urethral tissue of New Zealand White rabbits to examine biocompatibility in vivo. BPG concentrations of 50 mg/mL (n = 6, 1 week) and 200 mg/mL (n = 6, 1 week and n = 6, 6 weeks) dissolved in 1× phosphate-buffered saline (PBS) were injected transurethrally using a 9 French cystoscope, and were compared to PBS-injected controls (n = 3, 1 week) and non-injected controls (n = 2, 1 week). Urethral compression pressure measurements confirm BPG injections did not modify normal urethral pressure, as intended. Histological assessment demonstrated biological tolerance of BPGs in urethra and no inflammatory response was detected after 1 and 6 weeks compared to non-injected controls. Confocal imaging of fluorescently-labeled BPG injected urethral specimens demonstrated the integration of BPGs into the interstitial connective tissue and confirmed they were still present after 6 weeks. A general decrease of collagen density was exhibited near injection sites which may be due to increased hydration induced by BPGs. Injection of BPGs is a novel approach that demonstrates potential as molecular treatment for SUI and may be able to reverse some of the degenerative tissue changes of individuals affected by this condition. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: 00B: 000-000, 2019. © 2019 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 2409-2418, 2019.

Biomimetic proteoglycans can molecularly engineer early osteoarthritic cartilage in vivo.

Biomimetic proteoglycans (BPGs) have the potential to treat osteoarthritis (OA) given that these molecules mimic the structure and properties of natural proteoglycans, which are significantly reduced in OA. We examined the effects of BPGs injected into the intra-articular space in an in vivo OA rabbit knee model and evaluated the effect on histological response, joint friction, and BPG distribution and retention. Rabbits underwent ACL transection to create an arthritic state after 5 weeks. OA rabbits were treated with BPGs or Euflexxa® (hyaluronic acid) intra-articular injections. Non-OA rabbits were injected similarly with BPGs; contralateral joints served as controls. The progression of OA and response to injections were evaluated using Mankin and gross grading systems indicating that mild OA was achieved in operated joints. The coefficient of friction (COF) of the intact knee joints were measured using a custom pendulum friction apparatus, showing that OA joints and OA + Euflexxa® joints demonstrated increased COF than non-operated controls, while BPG-injected non-OA and OA + BPGs were not significantly different from non-OA controls. Injected fluorescently labeled BPGs demonstrated that BPGs diffused into cartilage with localization in the pericellular region. © 2018 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:403-411, 2019.

Electrospun poly(ε-caprolactone) nanofiber shish kebabs mimic mineralized bony surface features.

Electrospinning of nanofiber is of growing interest especially in bone tissue engineering because of its similar fibrous properties to the extracellular matrix. To this end, we have fabricated polycaprolactone (PCL) nanofiber shish kebab (NFSK) templates. The novelty of this work is the ability to control the mineral orientation and spatial location on the nanofiber, mimicking natural collagen fibers. However, NFSK templates have properties that need to be investigated in terms of cellular response including fiber alignment and crystallization. In this study, MC3T3 E1 preosteoblast cells were seeded onto the templates to determine the effect of both fiber orientation and kebab size on the cell metabolic activity. PCL was electrospun to form aligned and randomly oriented nanofibers, which were then crystallized in a PCL solution in pentyl acetate for 15 and 60 min, resulting in the formation of homopolymer PCL NFSK templates. We evaluated the cell proliferation and alkaline phosphatase activity of MC3T3 E1 cells after 3, 7, and 14 days in coculture. Aligned nanofiber and polymer crystallization both significantly increased the cell proliferation and alkaline phosphatase activity at each time point. The aligned nanofibers and polymer crystallization resulted in the highest metabolic activities of the cells compared to the randomly oriented fibers and noncrystallized controls. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1141-1149, 2019.

Structure-Property Relationships for 3D printed PEEK Intervertebral Lumbar Cages Produced using Fused Filament Fabrication.

Recent advances in additive manufacturing technology now enable fused filament fabrication (FFF) of Polyetheretherketone (PEEK). A standardized lumbar fusion cage design was 3D printed with different speeds of the print head nozzle to investigate whether 3D printed PEEK cages exhibit sufficient material properties for lumbar fusion applications. It was observed that the compressive and shear strength of the 3D printed cages were 63-71% of the machined cages, whereas the torsion strength was 92%. Printing speed is an important printing parameter for 3D printed PEEK, which resulted in up to 20% porosity at the highest speed of 3000 mm/min, leading to reduced cage strength. Printing speeds below 1500 mm/min can be chosen as the optimal printing speed for this printer to reduce the printing time while maintaining strength. The crystallinity of printed PEEK did not differ significantly from as-machined PEEK cages from extruded rods, indicating that the processing provides similar microstructure.

Translating Mechanobiology to the Clinic: a panel discussion from the 2018 CMBE Conference.

The 2018 BMES Cellular and Molecular Bioengineering (CMBE) Conference was organized around the theme of Discovering the Keys: Transformative and Translational Mechanobiology. The conference programing included a panel discussion on Translating Mechanobiology to the Clinic. The goal of the panel was to initiate a dialog and share pearls of wisdom from participants' successes and failures in academia and in industry toward translating scientific discoveries in the field of mechanobiology to technology products in the market or toward devices or drugs that impact clinical care. This commentary reviews the major themes and questions discussed during the panel, including defining translational research and how it applies to mechanobiology, the current landscape in translational mechanobiology, the process for translating mechanobiology research, challenges in translating mechanobiology research, and unique opportunities in translating mechanobiology research.