Promoting Bone Formation and Healing in Segmental Defects Through Ectopic Induced Membrane.

We aimed to determine whether addition of an in vivo ectopic induced membrane (EM) to the Masquelet Technique enhanced angiogenesis and bone formation in a segmental defect. After generating and stabilizing a diaphyseal femur defect, 10 rats received a polymethylmethacrylate (PMMA) spacer within the defect (control); 10 received another PMMA spacer implanted subcutaneously (EM). We removed the spacers and added autograft; the excised EM was added to their autograft (EM group). Post-mortem x-rays assessed bone formation and bridging. Osteogenesis in the proximal defect was significantly more uniform (p < 0.01), and there was greater amount of bone remodeling distally in the EM group (p < 0.05). There was no difference in bone formation (p = 0.19) but greater degrees of bridging in the EM group (2.20 vs. 1.20, p = 0.09). The EM resulted in more homogeneous proximal osteogenesis and increased bone remodeling distally. These findings could lead to more consistent and predictable bone healing. (Journal of Surgical Orthopaedic Advances 31(3):161-165, 2022).

Orthopaedic Implant Coatings: Recent Approaches and Clinical Translation.

Despite improved surgical techniques and prophylactic procedures, orthopaedic implant-associated infections remain high with complications that can lead to devastating outcomes for the patient. Implant coatings and associated surface modification techniques represent a promising means to prevent infections. Various approaches have emerged to address the challenges associated with implant infections, such as antibacterial resistance, biofilm prevention, and appropriate efficacy kinetics. Methods including antibiotic and antimicrobial peptide surface tethering, use of osteo-conductive and -inductive materials, and altering hydrophobicity and hydrophilicity of the implant surface, have all demonstrated efficacy toward diminished infection risk. Though many of these techniques have shown great potential in in vitro and in vivo studies, clinical translation remains limited with very few commercially available implant coatings globally. This review summarizes recent advancements in orthopaedic implant coatings, pre-clinical studies, and clinical translation, as well as potential future marketed products. (Journal of Surgical Orthopaedic Advances 31(3):169-176, 2022).

Control of adhesive ligand density for modulation of nucleus pulposus cell phenotype.

Cells of the nucleus pulposus have been observed to undergo a shift from their notochordal-like juvenile phenotype to a more fibroblast-like state with age and maturation. It has been demonstrated that culture of degenerative adult human nucleus pulposus cells upon soft (<1 kPa) full length laminin-containing hydrogel substrates promotes increased levels of a panel of markers associated with the juvenile nucleus pulposus cell phenotype. In the current work, we observed an ability to use soft polymeric substrates functionalized with short laminin-mimetic peptide sequences to recapitulate the behaviors elicited by soft, full-length laminin containing materials. Furthermore, our work suggests an ability to mimic features of soft systems through control of peptide density upon stiffer substrates. Specifically, results suggest that stiffer polymer-peptide hydrogel substrates can be used to promote the expression of a more juvenile-like phenotype for cells of the nucleus pulposus by reducing adhesive ligand presentation. Here we show how polymer stiffness combined with adhesive ligand presentation can be controlled to be supportive of nucleus pulposus cell phenotype and biosynthesis.

Verteporfin treatment controls morphology, phenotype, and global gene expression for cells of the human nucleus pulposus.

Cells of the nucleus pulposus (NP) are essential contributors to extracellular matrix synthesis and function of the intervertebral disc. With age and degeneration, the NP becomes stiffer and more dehydrated, which is associated with a loss of phenotype and biosynthetic function for its resident NP cells. Also, with aging, the NP cell undergoes substantial morphological changes from a rounded shape with pronounced vacuoles in the neonate and juvenile, to one that is more flattened and spread with a loss of vacuoles. Here, we make use of the clinically relevant pharmacological treatment verteporfin (VP), previously identified as a disruptor of yes-associated protein-TEA domain family member-binding domain (TEAD) signaling, to promote morphological changes in adult human NP cells in order to study variations in gene expression related to differences in cell shape. Treatment of adult, degenerative human NP cells with VP caused a shift in morphology from a spread, fibroblastic-like shape to a rounded, clustered morphology with decreased transcriptional activity of TEAD and serum-response factor. These changes were accompanied by an increased expression of vacuoles, NP-specific gene markers, and biosynthetic activity. The contemporaneous observation of VP-induced changes in cell shape and prominent, time-dependent changes within the transcriptome of NP cells occurred over all timepoints in culture. Enriched gene sets with the transition to VP-induced cell rounding suggest a major role for cell adhesion, cytoskeletal remodeling, vacuolar lumen, and MAPK activity in the NP phenotypic and functional response to changes in cell shape.

Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Cells of the adult nucleus pulposus (NP) are critically important in maintaining overall disc health and function. NP cells reside in a soft, gelatinous matrix that dehydrates and becomes increasingly fibrotic with age. Such changes result in physical cues of matrix stiffness that may be potent regulators of NP cell phenotype and may contribute to a transition toward a senescent and fibroblastic NP cell with a limited capacity for repair. Here, we investigate the mechanosignaling cues generated from changes in matrix stiffness in directing NP cell phenotype and identify mechanisms that can potentially preserve a biosynthetically active, juvenile NP cell phenotype. Using a laminin-functionalized polyethylene glycol hydrogel, we show that when NP cells form rounded, multicell clusters, they are able to maintain cytosolic localization of myocardin-related transcription factor (MRTF)-A, a coactivator of serum-response factor (SRF), known to promote fibroblast-like behaviors in many cells. Upon preservation of a rounded shape, human NP cells similarly showed cytosolic retention of transcriptional coactivator Yes-associated protein (YAP) and its paralogue PDZ-binding motif (TAZ) with associated decline in activation of its transcription factor TEA domain family member-binding domain (TEAD). When changes in cell shape occur, leading to a more spread, fibrotic morphology associated with stronger F-actin alignment, SRF and TEAD are up-regulated. However, targeted deletion of either cofactor was not sufficient to overcome shape-mediated changes observed in transcriptional activation of SRF or TEAD. Findings show that substrate stiffness-induced promotion of F-actin alignment occurs concomitantly with a flattened, spread morphology, decreased NP marker expression, and reduced biosynthetic activity. This work indicates cell shape is a stronger indicator of SRF and TEAD mechanosignaling pathways than coactivators MRTF-A and YAP/TAZ, respectively, and may play a role in the degeneration-associated loss of NP cellularity and phenotype.-Fearing, B. V., Jing, L., Barcellona, M. N., Witte, S. E., Buchowski, J. M., Zebala, L. P., Kelly, M. P., Luhmann, S., Gupta, M. C., Pathak, A., Setton, L. A. Mechanosensitive transcriptional coactivators MRTF-A and YAP/TAZ regulate nucleus pulposus cell phenotype through cell shape.

Mechanotransduction and cell biomechanics of the intervertebral disc.

Mechanical loading of the intervertebral disc (IVD) initiates cell-mediated remodeling events that contribute to disc degeneration. Cells of the IVD, nucleus pulposus (NP) and anulus fibrosus (AF), will exhibit various responses to different mechanical stimuli which appear to be highly dependent on loading type, magnitude, duration, and anatomic zone of cell origin. Cells of the NP, the innermost region of the disc, exhibit an anabolic response to low-moderate magnitudes of static compression, osmotic pressure, or hydrostatic pressure, while higher magnitudes promote a catabolic response marked by increased protease expression and activity. Cells of the outer AF are responsive to physical forces in a manner that depends on frequency and magnitude, as are cells of the NP, though they experience different forces, deformations, pressure, and osmotic pressure in vivo. Much remains to be understood of the mechanotransduction pathways that regulate IVD cell responses to loading, including responses to specific stimuli and also differences among cell types. There is evidence that cytoskeletal remodeling and receptor-mediated signaling are important mechanotransduction events that can regulate downstream effects like gene expression and posttranslational biosynthesis, all of which may influence phenotype and bioactivity. These and other mechanotransduction events will be regulated by known and to-be-discovered cell-matrix and cell-cell interactions, and depend on composition of extracellular matrix ligands for cell interaction, matrix stiffness, and the phenotype of the cells themselves. Here, we present a review of the current knowledge of the role of mechanical stimuli and the impact upon the cellular response to loading and changes that occur with aging and degeneration of the IVD.

Regulation of human nucleus pulposus cells by peptide-coupled substrates.

Nucleus pulposus (NP) cells are derived from the notochord and differ from neighboring cells of the intervertebral disc in phenotypic marker expression and morphology. Adult human NP cells lose this phenotype and morphology with age in a pattern that contributes to progressive disc degeneration and pathology. Select laminin-mimetic peptide ligands and substrate stiffnesses were examined for their ability to regulate human NP cell phenotype and biosynthesis through the expression of NP-specific markers aggrecan, N-cadherin, collagen types I and II, and GLUT1. Peptide-conjugated substrates demonstrated an ability to promote expression of healthy NP-specific markers, as well as increased biosynthetic activity. We show an ability to re-express markers of the juvenile NP cell and morphology through control of peptide presentation and stiffness on well-characterized polyacrylamide substrates. NP cells cultured on surfaces conjugated with α3 integrin receptor peptides P4 and P678, and on α2, α5, α6, ß1 integrin-recognizing peptide AG10, show increased expression of aggrecan, N-cadherin, and types I and II collagen, suggesting a healthier, more juvenile-like phenotype. Multi-cell cluster formation was also observed to be more prominent on peptide-conjugated substrates. These findings indicate a critical role for cell-matrix interactions with specific ECM-mimetic peptides in supporting and maintaining a healthy NP cell phenotype and bioactivity. STATEMENT OF SIGNIFICANCE: NP cells reside in a laminin-rich environment that deteriorates with age, including a loss of water content and changes in the extracellular matrix (ECM) structure that may lead to the development of a degenerated IVD. There is great interest in methods to re-express healthy, biosynthetically active NP cells using laminin-derived biomimetic peptides toward the goal of using autologous cell sources for tissue regeneration. Here, we describe a novel study utilizing several laminin mimetic peptides conjugated to polyacrylamide gels that are able to support an immature, healthy NP phenotype after culture on "soft" peptide gels. These findings can support future studies in tissue regeneration where cells may be directed to a desired regenerative phenotype using niche-specific ECM peptides.

In vitro response of macrophage polarization to a keratin biomaterial.

Macrophage response to biomaterials is emerging as a major focus in tissue repair and wound healing. Macrophages are able to differentiate into two distinct states, eliciting divergent effects. The M1 phenotype is considered pro-inflammatory and up-regulates activity related to tissue destruction, whereas the M2 phenotype is considered anti-inflammatory and supports tissue remodeling. Both are necessary but a fine balance must be maintained as dysregulation of naïve macrophages to M1 or M2 polarization has been implicated in several disease and injury models, and has been suggested as a potential cause for poor outcomes. Keratin biomaterials have been shown using different animal models to promote regeneration in several tissues. A potential common mechanism may be the general capability for keratin biomaterials to elicit beneficial inflammatory responses during the early stages of regeneration. In the present study, a keratin biomaterial was utilized in vitro to examine its effects on polarization toward one of these two macrophage phenotypes, and thus its role in inflammation. Exposure of a monocytic cell line to keratin biomaterial substrates was shown to bias macrophages toward an M2 phenotype, while a collagen control surface produced both M1 and M2 macrophages. Furthermore, keratin treatment was similar to the M2 positive control and was similarly effective at down-regulating the M1 response. Keratin biomaterial influenced greater production of anti-inflammatory cytokines and decreased amounts of pro-inflammatory cytokines. The use of a keratin biomaterial in regenerative medicine may therefore provide additional benefit by regulating a positive remodeling response.