Biofabrication Approaches With Hyaluronic Acid Hydrogels For Cartilage Repair

Current therapies to repair damaged articular cartilage fail to consistently or fully restore the biomechanical function of cartilage. Although cell-based clinical techniques have emerged for the treatment of focal defects in articulating joints, these approaches typically lead to inferior tissue formation when compared to native, healthy cartilage. Alternatively, subchondral microfracture is a surgical procedure that aims to recruit endogenous mesenchymal stromal cells (MSCs) from the underlying bone marrow to facilitate neocartilage formation in focal defects. Similarly, microfracture typically results in the formation of repair cartilage incapable of withstanding the loading environment of the articulating joint over time. New biomaterial-based strategies are therefore in significant demand to improve cartilage tissue formation and maturation within focal defects. Hyaluronic acid (HA) is a glycosaminoglycan that is found in native cartilage and that shows promise as a biomaterial for cartilage tissue engineering due to its innate bioactivity and ability to form hydrogels, water-swollen polymer networks that may be engineered to mimic the native extracellular matrix (ECM). Moreover, hydrogels may be employed as materials for biofabrication, which involves the use of automated additive manufacturing processes such as 3D printing to fabricate living, biological constructs. This dissertation describes the design and implementation of HA hydrogels for the biofabrication of articular cartilage towards improving existing therapies for damaged cartilage. Multiple biofabrication approaches, including extrusion bioprinting, melt-electrowriting, and digital light processing are investigated to engineer scaffolds with rationally designed geometries, mechanical properties, porosities, and biodegradability. Conserved across all these approaches is the use of thiol-ene based photochemistry to control the formation and resultant material properties of HA hydrogels modified with norbornene functional groups. Taken together, the employment of these biofabrication approaches for cartilage repair has significantly informed the design and implementation of future therapies for articular cartilage damage

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage

Integrated HIV Testing, Malaria, and Diarrhea Prevention Campaign in Kenya: Modeled Health Impact and Cost-Effectiveness

Efficiently delivered interventions to reduce HIV, malaria, and diarrhea are essential to accelerating global health efforts. A 2008 community integrated prevention campaign in Western Province, Kenya, reached 47,000 individuals over 7 days, providing HIV testing and counseling, water filters, insecticide-treated bed nets, condoms, and for HIV-infected individuals cotrimoxazole prophylaxis and referral for ongoing care. We modeled the potential cost-effectiveness of a scaled-up integrated prevention campaign.We estimated averted deaths and disability-adjusted life years (DALYs) based on published data on baseline mortality and morbidity and on the protective effect of interventions, including antiretroviral therapy. We incorporate a previously estimated scaled-up campaign cost. We used published costs of medical care to estimate savings from averted illness (for all three diseases) and the added costs of initiating treatment earlier in the course of HIV disease.Per 1000 participants, projected reductions in cases of diarrhea, malaria, and HIV infection avert an estimated 16.3 deaths, 359 DALYs and $85,113 in medical care costs. Earlier care for HIV-infected persons adds an estimated 82 DALYs averted (to a total of 442), at a cost of$37,097 (reducing total averted costs to $48,015). Accounting for the estimated campaign cost of$32,000, the campaign saves an estimated $16,015 per 1000 participants. In multivariate sensitivity analyses, 83$20.A mass, rapidly implemented campaign for HIV testing, safe water, and malaria control appears economically attractive

Biofabrication Approaches with Hyaluronic Acid Hydrogels for Cartilage Repair

Current therapies to repair damaged articular cartilage fail to consistently or fully restore the biomechanical function of cartilage. Although cell-based clinical techniques have emerged for the treatment of focal defects in articulating joints, these approaches typically lead to inferior tissue formation when compared to native, healthy cartilage. Alternatively, subchondral microfracture is a surgical procedure that aims to recruit endogenous mesenchymal stromal cells (MSCs) from the underlying bone marrow to facilitate neocartilage formation in focal defects. Similarly, microfracture typically results in the formation of repair cartilage incapable of withstanding the loading environment of the articulating joint over time. New biomaterial-based strategies are therefore in significant demand to improve cartilage tissue formation and maturation within focal defects. Hyaluronic acid (HA) is a glycosaminoglycan that is found in native cartilage and that shows promise as a biomaterial for cartilage tissue engineering due to its innate bioactivity and ability to form hydrogels, water-swollen polymer networks that may be engineered to mimic the native extracellular matrix (ECM). Moreover, hydrogels may be employed as materials for biofabrication, which involves the use of automated additive manufacturing processes such as 3D printing to fabricate living, biological constructs. This dissertation describes the design and implementation of HA hydrogels for the biofabrication of articular cartilage towards improving existing therapies for damaged cartilage. Multiple biofabrication approaches, including extrusion bioprinting, melt-electrowriting, and digital light processing are investigated to engineer scaffolds with rationally designed geometries, mechanical properties, porosities, and biodegradability. Conserved across all these approaches is the use of thiol-ene based photochemistry to control the formation and resultant material properties of HA hydrogels modified with norbornene functional groups. Taken together, the employment of these biofabrication approaches for cartilage repair has significantly informed the design and implementation of future therapies for articular cartilage damage

Fabrication of MSC-laden composites of hyaluronic acid hydrogels reinforced with MEW scaffolds for cartilage repair

Hydrogels are of interest in cartilage tissue engineering due to their ability to support the encapsulation and chondrogenesis of mesenchymal stromal cells (MSCs). However, features such as hydrogel crosslink density, which can influence nutrient transport, nascent matrix distribution, and the stability of constructs during and after implantation must be considered in hydrogel design. Here, we first demonstrate that more loosely crosslinked (i.e. softer, ∼2 kPa) norbornene-modified hyaluronic acid (NorHA) hydrogels support enhanced cartilage formation and maturation when compared to more densely crosslinked (i.e. stiffer, ∼6-60 kPa) hydrogels, with a >100-fold increase in compressive modulus after 56 d of culture. While soft NorHA hydrogels mature into neocartilage suitable for the repair of articular cartilage, their initial moduli are too low for handling and they do not exhibit the requisite stability needed to withstand the loading environments of articulating joints. To address this, we reinforced NorHA hydrogels with polycaprolactone (PCL) microfibers produced via melt-electrowriting (MEW). Importantly, composites fabricated with MEW meshes of 400 µm spacing increased the moduli of soft NorHA hydrogels by ∼50-fold while preserving the chondrogenic potential of the hydrogels. There were minimal differences in chondrogenic gene expression and biochemical content (e.g. DNA, GAG, collagen) between hydrogels alone and composites, whereas the composites increased in compressive modulus to ∼350 kPa after 56 d of culture. Lastly, integration of composites with native tissue was assessed ex vivo; MSC-laden composites implanted after 28 d of pre-culture exhibited increased integration strengths and contact areas compared to acellular composites. This approach has great potential towards the design of cell-laden implants that possess both initial mechanical integrity and the ability to support neocartilage formation and integration for cartilage repair

Fabrication of MSC-laden composites of hyaluronic acid hydrogels reinforced with MEW scaffolds for cartilage repair

Hydrogels are of interest in cartilage tissue engineering due to their ability to support the encapsulation and chondrogenesis of mesenchymal stromal cells (MSCs). However, features such as hydrogel crosslink density, which can influence nutrient transport, nascent matrix distribution, and the stability of constructs during and after implantation must be considered in hydrogel design. Here, we first demonstrate that more loosely crosslinked (i.e. softer, ∼2 kPa) norbornene-modified hyaluronic acid (NorHA) hydrogels support enhanced cartilage formation and maturation when compared to more densely crosslinked (i.e. stiffer, ∼6-60 kPa) hydrogels, with a >100-fold increase in compressive modulus after 56 d of culture. While soft NorHA hydrogels mature into neocartilage suitable for the repair of articular cartilage, their initial moduli are too low for handling and they do not exhibit the requisite stability needed to withstand the loading environments of articulating joints. To address this, we reinforced NorHA hydrogels with polycaprolactone (PCL) microfibers produced via melt-electrowriting (MEW). Importantly, composites fabricated with MEW meshes of 400µm spacing increased the moduli of soft NorHA hydrogels by ∼50-fold while preserving the chondrogenic potential of the hydrogels. There were minimal differences in chondrogenic gene expression and biochemical content (e.g. DNA, GAG, collagen) between hydrogels alone and composites, whereas the composites increased in compressive modulus to ∼350 kPa after 56 d of culture. Lastly, integration of composites with native tissue was assessedex vivo; MSC-laden composites implanted after 28 d of pre-culture exhibited increased integration strengths and contact areas compared to acellular composites. This approach has great potential towards the design of cell-laden implants that possess both initial mechanical integrity and the ability to support neocartilage formation and integration for cartilage repair

Evaluation of surgical fixation methods for the implantation of melt electrowriting-reinforced hyaluronic acid hydrogel composites in porcine cartilage defects

The surgical repair of articular cartilage remains an ongoing challenge in orthopedics. Tissue engineering is a promising approach to treat cartilage defects; however, scaffolds must (i) possess the requisite material properties to support neocartilage formation, (ii) exhibit sufficient mechanical integrity for handling during implantation, and (iii) be reliably fixed within cartilage defects during surgery. In this study, we demonstrate the reinforcement of soft norbornene-modified hyaluronic acid (NorHA) hydrogels via the melt electrowriting (MEW) of polycaprolactone to fabricate composite scaffolds that support encapsulated porcine mesenchymal stromal cell (pMSC, three donors) chondrogenesis and cartilage formation and exhibit mechanical properties suitable for handling during implantation. Thereafter, acellular MEW-NorHA composites or MEW-NorHA composites with encapsulated pMSCs and precultured for 28 days were implanted in full-thickness cartilage defects in porcine knees using either bioresorbable pins or fibrin glue to assess surgical fixation methods. Fixation of composites with either biodegradable pins or fibrin glue ensured implant retention in most cases (80%); however, defects treated with pinned composites exhibited more subchondral bone remodeling and inferior cartilage repair, as evidenced by micro-computed tomography (micro-CT) and safranin O/fast green staining, respectively, when compared to defects treated with glued composites. Interestingly, no differences in repair tissue were observed between acellular and cellularized implants. Additional work is required to assess the full potential of these scaffolds for cartilage repair. However, these results suggest that future approaches for cartilage repair with MEW-reinforced hydrogels should be carefully evaluated with regard to their fixation approach for construct retention and surrounding cartilage tissue damage.</p

Rational use of antiretroviral therapy in low-income and middle-income countries: optimizing regimen sequencing and switching

During the 4 years to the end of 2007, the number of people in low-income and middle-income countries (LMICs) receiving antiretroviral therapy (ART) increased from 400 000 to 3 million [1,2]. Although early mortality [3] and retention in care [4] remain significant challenges, the majority of reports from LMICs have shown encouraging immunological, virological and survival outcomes [5 12]. Reported rates of switching to second-line ART regimens have been lower than expected [13 15], in part due to actual rates of treatment success, but mainly because of limited access to both virological monitoring [16] and second-line drugs [14]. Clinicians have also been reluctant to switch therapy [15] due to regimen cost, complexity, inconvenience and lack of subsequent treatment options. As cohorts mature and expand and access to virological monitoring and second-line regimens increase, however, rates of diagnosed treatment failure and switch to second-line regimens will increase [17]. As the cost of second-line regimens are currently three to 20 times more expensive than that of first-line regimens [18], these increases will challenge the cost-effectiveness [19,20] and sustainability [21] of HIV-treatment programmes.An effective response to the challenges of HIV treatment failure in LMICs must include reductions in the cost of second-line agents [22], but changes to commercial regulations, particularly in India, suggest the scale of price reductions seen with first-line agents are unlikely to occur with second-line agents. Strategies to maximize the effectiveness of first-line and second-line regimens and optimize the timing of regimen switching are required to fully utilize the survival benefit of available treatment options, maintain programme cost-effectiveness and enable achievement of universal access to HIV treatment. A comprehensive strategy must be evidence based and focused on the rational long-term use of ART at a population level. The objective of this review is to support the development of these strategies by providing an overview of available evidence with an emphasis on regimen sequencing and switching