Semi-synthetic degradable notochordal cell-derived matrix hydrogel for use in degenerated intervertebral discs: Initial in vitro characterization.

Low back pain is the leading cause of disability worldwide, but current therapeutic interventions are palliative or surgical in nature. Loss of notochordal cells (NCs) and degradation of the healthy matrix in the nucleus pulposus (NP), the central tissue of intervertebral discs (IVDs), has been associated with onset of degenerative disc changes. Recently, we established a protocol for decellularization of notochordal cell derived matrix (NCM) and found that it can provide regenerative cues to nucleus pulposus cells of the IVD. Here, we combined the biologically regenerative properties of decellularized NCM with the mechanical tunability of a poly(ethylene glycol) hydrogel to additionally address biomechanics in the degenerate IVD. We further introduced a hydrolysable PEG-diurethane crosslinker for slow degradation of the gels in vivo. The resulting hydrogels were tunable over a broad range of stiffness's (0.2 to 4.5 kPa), matching that of NC-rich and -poor NP tissues, respectively. Gels formed within 30 min, giving ample time for handling, and remained shear-thinning post-polymerization. Gels also slowly released dNCM over 28 days as measured by GAG effusion. Viability of encapsulated bone marrow stromal cells after extrusion through a needle remained high. Although encapsulated NCs stayed viable over two weeks, their metabolic activity decreased, and their phenotype was lost in physiological medium conditions in vitro. Overall, the obtained gels hold promise for application in degenerated IVDs but require further tuning for combined use with NCs.

Harmonization and standardization of nucleus pulposus cell extraction and culture methods.

Background: In vitro studies using nucleus pulposus (NP) cells are commonly used to investigate disc cell biology and pathogenesis, or to aid in the development of new therapies. However, lab-to-lab variability jeopardizes the much-needed progress in the field. Here, an international group of spine scientists collaborated to standardize extraction and expansion techniques for NP cells to reduce variability, improve comparability between labs and improve utilization of funding and resources. Methods: The most commonly applied methods for NP cell extraction, expansion, and re-differentiation were identified using a questionnaire to research groups worldwide. NP cell extraction methods from rat, rabbit, pig, dog, cow, and human NP tissue were experimentally assessed. Expansion and re-differentiation media and techniques were also investigated. Results: Recommended protocols are provided for extraction, expansion, and re-differentiation of NP cells from common species utilized for NP cell culture. Conclusions: This international, multilab and multispecies study identified cell extraction methods for greater cell yield and fewer gene expression changes by applying species-specific pronase usage, 60-100 U/ml collagenase for shorter durations. Recommendations for NP cell expansion, passage number, and many factors driving successful cell culture in different species are also addressed to support harmonization, rigor, and cross-lab comparisons on NP cells worldwide.

The loop of phenotype: Dynamic reciprocity links tenocyte morphology to tendon tissue homeostasis.

Cells and their surrounding extracellular matrix (ECM) are engaged in dynamic reciprocity to maintain tissue homeostasis: cells deposit ECM, which in turn presents the signals that define cell identity. This loop of phenotype is obvious for biochemical signals, such as collagens, which are produced by and presented to cells, but the role of biomechanical signals is also increasingly recognised. In addition, cell shape goes hand in hand with cell function and tissue homeostasis. Aberrant cell shape and ECM is seen in pathological conditions, and control of cell shape in micro-fabricated platforms disclose the causal relationship between cell shape and cell function, often mediated by mechanotransduction. In this manuscript, we discuss the loop of phenotype for tendon tissue homeostasis. We describe cell shape and ECM organization in normal and diseased tissue, how ECM composition influences tenocyte shape, and how that leads to the activation of signal transduction pathways and ECM deposition. We further describe the use of technologies to control cell shape to elucidate the link between cell shape and its phenotypical markers and focus on the causal role of cell shape in the loop of phenotype. STATEMENT OF SIGNIFICANCE: The dynamic reciprocity between cells and their surrounding extracellular matrix (ECM) influences biomechanical and biochemical properties of ECM as well as cell function through activation of signal transduction pathways that regulate gene and protein expression. We refer to this reciprocity as Loop of Phenotype and it has been studied and demonstrated extensively by using micro-fabricated platforms to manipulate cell shape and cell fate. In this manuscript, we discuss this concept in tendon tissue homeostasis by giving examples in healthy and pathological tenson tissue. Furthermore, we elaborate this by showing how biomaterials are used to feed this reciprocity to manipulate cell shape and function. Finally, we elucidate the link between cell shape and its phenotypical markers and focus on the activation of signal transduction pathways and ECM deposition.

Detergent-Free Decellularization of Notochordal Cell-Derived Matrix Yields a Regenerative, Injectable, and Swellable Biomaterial.

Porcine notochordal cell-derived matrix (NCM) has anti-inflammatory and regenerative effects on degenerated intervertebral discs. For its clinical use, safety must be assured. The porcine DNA is concerning because of (1) the transmission of endogenous retroviruses and (2) the inflammatory potential of cell-free DNA. Here, we present a simple, detergent-free protocol: tissue lyophilization lyses cells, and matrix integrity is preserved by limiting swelling during decellularization. DNA is digested quickly by a high nuclease concentration, followed by a short washout. Ninety-four percent of DNA was removed, and there was no loss of glycosaminoglycans or collagen. Forty-three percent of the total proteins remained in the decellularized NCM (dNCM). dNCM stimulated as much GAG production as NCM in nucleus pulposus cells but lost some anti-inflammatory effects. Reconstituted pulverized dNCM yielded a soft, shear-thinning biomaterial with a swelling ratio of 350% that also acted as an injectable cell carrier (cell viability >70%). dNCM can therefore be used as the basis for future biomaterials aimed at disc regeneration on a biological level and may restore joint mechanics by creating swelling pressure within the intervertebral disc.

Injectable Hydrogels for Articular Cartilage and Nucleus Pulposus Repair: Status Quo and Prospects.

Osteoarthritis (OA) and chronic low back pain due to degenerative (intervertebral) disc disease (DDD) are two of the major causes of disabilities worldwide, affecting hundreds of millions of people and leading to a high socioeconomic burden. Although OA occurs in synovial joints and DDD occurs in cartilaginous joints, the similarities are striking, with both joints showing commonalities in the nature of the tissues and in the degenerative processes during disease. Consequently, repair strategies for articular cartilage (AC) and nucleus pulposus (NP), the core of the intervertebral disc, in the context of OA and DDD share common aspects. One of such tissue engineering approaches is the use of injectable hydrogels for AC and NP repair. In this review, the state-of-the-art and recent developments in injectable hydrogels for repairing, restoring, and regenerating AC tissue suffering from OA and NP tissue in DDD are summarized focusing on cell-free approaches. The various biomaterial strategies exploited for repair of both tissues are compared, and the synergies that could be gained by translating experiences from one tissue to the other are identified. Impact statement Joints affected by osteoarthritis (OA) and degenerative (intervertebral) disc disease (DDD) share similarities in tissue composition and in the degenerative disease processes. This has led to the development of similar tissue engineering approaches to repair the articular cartilage (AC) and the nucleus pulposus (NP), in the context of OA and DDD, such as injectable hydrogels. In this review, recent developments in injectable hydrogels for repair of AC and NP tissues are summarized, biomaterial strategies are compared, and synergies are identified focusing on cell-free approaches. The summarized developments are expected to inspire more cross talk between both research fields.

Magnetite-binding proteins from the magnetotactic bacterium Desulfamplus magnetovallimortis BW-1.

Magnetite-binding proteins are in high demand for the functionalization of magnetic nanoparticles. Binding analysis of six previously uncharacterized proteins from the magnetotactic Deltaproteobacterium Desulfamplus magnetovallimortis BW-1 identified two new magnetite-binding proteins (Mad10, Mad11). These proteins can be utilized as affinity tags for the immobilization of recombinant fusion proteins to magnetite.

Solid-phase silica-based extraction leads to underestimation of residual DNA in decellularized tissues.

Decellularization of animal tissues is a novel route to obtain biomaterials for use in tissue engineering and organ transplantation. Successful decellularization is required as animal DNA causes inflammatory reactions and contains endogenous retroviruses, which could be transmitted to the patient. One of the criteria for successful decellularization is digestion (fragmentation) and elimination (residual quantity) of DNA from the tissue. Quantification of DNA can be done in many ways, but it has recently been shown that silica-based solid-phase extraction methods often do not completely purify in particular small DNA fragments. In the context of decellularization, this means that the measured DNA amount is underestimated, which could compromise safety of the processed tissue for in-patient use. In this article, we review DNA quantification methods used by researchers and assess their influence on the reported DNA contents after decellularization. We find that underestimation of residual DNA amount after silica-based solid-phase extraction may be as large as a factor of ten. We therefore recommend a direct assessment of DNA amount in tissue lysate using dsDNA-specific binding dyes, such as Picogreen, due to their higher accuracy for small fragment detection as well as ease of use and widespread availability.

Characterization of biomaterials intended for use in the nucleus pulposus of degenerated intervertebral discs.

Biomaterials for regeneration of the intervertebral disc must meet complex requirements conforming to biological, mechanical and clinical demands. Currently no consensus on their characterization exists. It is crucial to identify parameters and their method of characterization for accurate assessment of their potential efficacy, keeping in mind the translation towards clinical application. This review systematically analyses the characterization techniques of biomaterial systems that have been used for nucleus pulposus (NP) restoration and regeneration. Substantial differences in the approach towards assessment became evident, hindering comparisons between different materials with respect to their suitability for NP restoration and regeneration. We have analysed the current approaches and identified parameters necessary for adequate biomaterial characterization, with the clinical goal of functional restoration and biological regeneration of the NP in mind. Further, we provide guidelines and goals for their measurement. STATEMENT OF SIGNIFICANCE: Biomaterials intended for restoration of regeneration of the nucleus pulposus within the intervertebral disc must meet biological, biomechanical and clinical demands. Many materials have been investigated, but a lack of consensus on which parameters to evaluate leads to difficulties in comparing materials as well as mostly partial characterization of the materials in question. A gap between current methodology and clinically relevant and meaningful characterization is prevalent. In this article, we identify necessary methods and their implementation for complete biomaterial characterization in the context of clinical applicability. This will allow for a more unified approach to NP-biomaterials research within the field as a whole and enable comparative analysis of novel materials yet to be developed.

Is Telehealth Right for Your Practice and Your Patients With Asthma?

INTRODUCTION: Telehealth services are becoming increasingly used to provide care to patients living in rural areas. Little is known about the patient satisfaction with the provision of these services. METHODS: A prospective cohort pilot study was developed to evaluate the use of telehealth for the delivery of asthma education services in the rural, medically underserved community of Oakes, North Dakota. A certified asthma educator used real-time, audio-visual telehealth technology to meet with patients the local community pharmacy. Patients met with the educator monthly for the first three months of the study, and once every three months thereafter. Patient satisfaction was measured using a five item survey. RESULTS: Eighteen patients completed the study (90 percent completion rate). Patient satisfaction scores were relatively high, typically between 4 and 5 on a 5-point scale. CONCLUSIONS: Participants in a rural, medically underserved community found the community pharmacy location and the telehealth technology a convenient means to access a specialty provider for asthma education.

Impact of telepharmacy services as a way to increase access to asthma care.

OBJECTIVE: To demonstrate that real-time, telepharmacy-based asthma educational services are feasible and to support the efforts of local primary care prescribers to improve patient outcomes. METHODS: The lead investigator (a pharmacist, physician assistant, and certified asthma educator) identified an independent community pharmacy with telehealth capabilities in a rural area with a high prevalence of asthma. Working with the pharmacy, an asthma education program was developed based on the National Asthma Education and Prevention Program guidelines. It consisted of three monthly education visits, with subsequent visits every three months for one year. The Asthma Control Test (ACT) was administered at baseline and at each visit to assess a patient's perception of asthma control. RESULTS: Eighteen of 20 patients (90%) with reversible airway disease completed all six visits in this year-long study. For the 18 patients, the mean ACT scores of 18 at baseline (initiation of intervention) did not meet the threshold for "well-controlled" asthma. By the third educational visit (3 months), 16 patients met ACT criteria for well-controlled asthma (mean score = 20), and they maintained control for the remaining 9-month follow-up period (ACT ≥ 21). Local prescribers authorized medication changes recommended by the asthma educator 20 times and also requested six direct consults with the asthma educator over the study period. CONCLUSION: Using the local community pharmacy as a vehicle to deliver asthma education services by telepharmacy was utilized by local prescribers. The findings show this is an effective means to engage patients to gain and maintain asthma control.

Performance and Perceptions: Evaluation of Pharmacy Students' Consultation via Telepharmacy.

Background: Pharmacy education standards highlight the importance of effective communication skills and the use of technology to provide patient care. As technology evolves, pharmacists have opportunities to communicate in different and broader ways. Objective: The objectives of this study were 3-fold: to evaluate student ability to counsel via telepharmacy, to determine if there is a difference in students' abilities to counsel face-to-face or via telepharmacy, and to determine students' perceptions regarding patient consultation via telepharmacy. Methods: Professional pharmacy students completed a pharmaceutical care laboratory activity focused on communication via telepharmacy. Comparisons were made between students' ability to provide patient consultation via telepharmacy and face-to-face utilizing a faculty-developed rubric. Students also completed a questionnaire on their perception of utilizing telepharmacy technology to provide patient consultation. Results: Eighty-two second-year professional pharmacy students participated in the study. Results showed students are able to successfully provide patient consultation via telepharmacy without prior practice; however, there was a statistically significant difference between students' ability to counsel face-to-face and via telepharmacy (P < .001). Overall, students were more successful at providing face-to-face consultation than via telepharmacy, and students who were first assessed on their ability to counsel face-to-face perceived a greater difference between telepharmacy and face-to-face consultation (P < .05). Conclusion: Student-perceived differences between the 2 means of consultation and demonstrated a difference in their ability to counsel via telepharmacy and face-to-face. It appears that, when evaluating the need to teach professional pharmacy students how to provide patient consultation via telepharmacy, additional exposure to telepharmacy technology could be beneficial by enhancing student comfort and proficiency.