Richard A. Hopkins

Decellularized cartilage may be a chondroinductive material for osteochondral tissue engineering.

Extracellular matrix (ECM)-based materials are attractive for regenerative medicine in their ability to potentially aid in stem cell recruitment, infiltration, and differentiation without added biological factors. In musculoskeletal tissue engineering, demineralized bone matrix is widely used, but recently cartilage matrix has been attracting attention as a potentially chondroinductive material. The aim of this study was thus to establish a chemical decellularization method for use with articular cartilage to quantify removal of cells and analyze the cartilage biochemical content at various stages during the decellularization process, which included a physically devitalization step. To study the cellular response to the cartilage matrix, rat bone marrow-derived mesenchymal stem cells (rBMSCs) were cultured in cell pellets containing cells only (control), chondrogenic differentiation medium (TGF-β), chemically decellularized cartilage particles (DCC), or physically devitalized cartilage particles (DVC). The chemical decellularization process removed the vast majority of DNA and about half of the glycosaminoglycans (GAG) within the matrix, but had no significant effect on the amount of hydroxyproline. Most notably, the DCC group significantly outperformed TGF-β in chondroinduction of rBMSCs, with collagen II gene expression an order of magnitude or more higher. While DVC did not exhibit a chondrogenic response to the extent that DCC did, DVC had a greater down regulation of collagen I, collagen X and Runx2. A new protocol has been introduced for cartilage devitalization and decellularization in the current study, with evidence of chondroinductivity. Such bioactivity along with providing the ‘raw material’ building blocks of regenerating cartilage may suggest a promising role for DCC in biomaterials that rely on recruiting endogenous cell recruitment and differentiation for cartilage regeneration.

Performance and Morphology of Decellularized Pulmonary Valves Implanted in Juvenile Sheep

BACKGROUND Because of cryopreserved heart valve-mediated immune responses, decellularized allograft valves are an attractive option in children and young adults. The objective of this study was to investigate the performance and morphologic features of decellularized pulmonary valves implanted in the right ventricular outflow tract of juvenile sheep. METHODS Right ventricular outflow tract reconstructions in juvenile sheep (160±9 days) using cryopreserved pulmonary allografts (n=6), porcine aortic root bioprostheses (n=4), or detergent/enzyme-decellularized pulmonary allografts (n=8) were performed. Valve performance (echocardiography) and morphologic features (gross, radiographic, and histologic examination) were evaluated 20 weeks after implantation. RESULTS Decellularization reduced DNA in valve cusps by 99.3%. Bioprosthetic valves had the largest peak and mean gradients versus decellularized valves (p=0.03; p<0.001) and cryopreserved valves (p=0.01; p=0.001), which were similar (p=0.45; p=0.40). Regurgitation was minimal and similar for all groups (p=0.16). No cusp calcification was observed in any valve type. Arterial wall calcification was present in cryopreserved and bioprosthetic grafts but not in decellularized valves. No autologous recellularization or inflammation occurred in bioprostheses, whereas cellularity progressively decreased in cryopreserved grafts. Autologous recellularization was present in decellularized arterial walls and variably extending into the cusps. CONCLUSIONS Cryopreserved and decellularized graft hemodynamic performance was comparable. Autologous recellularization of the decellularized pulmonary arterial wall was consistently observed, with variable cusp recellularization. As demonstrated in this study, decellularized allograft valves have the potential for autologous recellularization.

Effects of cryopreservation, decellularization and novel extracellular matrix conditioning on the quasi-static and time-dependent properties of the pulmonary valve leaflet

Decellularized allografts offer potential as heart valve substitutes and scaffolds for cell seeding. The effects of decellularization on the quasi-static and time-dependent mechanical behavior of the pulmonary valve leaflet under biaxial loading conditions have not previously been reported in the literature. In the current study, the stress-strain, relaxation and creep behaviors of the ovine pulmonary valve leaflet were investigated under planar-biaxial loading conditions to determine the effects of decellularization and a novel post-decellularization extracellular matrix (ECM) conditioning process. As expected, decellularization resulted in increased stretch along the loading axes. A reduction in relaxation was observed following decellularization. This was accompanied by a reduction in glycosaminoglycan (GAG) content. Based on previous implant studies, these changes may be of little functional consequence in the short term; however, the long term effects of decreased relaxation and GAG content remain unknown. Some restoration of relaxation was observed following ECM conditioning, especially in the circumferential specimen direction, which may help mitigate any detrimental effects due to decellularization. Regardless of processing, creep under biaxial loading was negligible.

Ovine atadenovirus, a novel and highly immunogenic vector in prime-boost studies of a candidate HIV-1 vaccine.

Ovine adenovirus type 7 (OAdV) is the prototype member of the genus Atadenovirus. No immunity to the virus has so far been detected in human sera. We describe the construction and evaluation of a candidate HIV-1 vaccine based on OAdV and its utilisation alone and in combination with plasmid-, human adenovirus type 5 (HAdV5; a Mastadenovirus)-, and modified vaccinia Ankara (MVA)-vectored vaccines. All vectors expressed HIVA, an immunogen consisting of HIV-1 clade A consensus Gag-derived protein coupled to a T cell polyepitope. OAdV.HIVA was genetically stable, grew well and expressed high levels of protein from the Rous sarcoma virus promoter. OAdV.HIVA was highly immunogenic in mice and efficiently primed and boosted HIV-1-specific T cell responses together with heterologous HIVA-expressing vectors. There were significant differences between OAdV and HAdV5 vectors in priming of naïve CD8(+) T cell responses to HIVA and in the persistence of MHC class I-restricted epitope presentation in the local draining lymph nodes. OAdV.HIVA primed T cells more rapidly but was less persistent than AdV5.HIVA and thus induced a qualitatively distinct T cell response. Nevertheless, both vectors primed a response in mice that reduced viral titres in a surrogate challenge model by three to four orders of magnitude. Thus, OAdV is a novel, underexplored vaccine vector with potential for further development for HIV-1 and other vaccines. The data are discussed in the context of the latest HIV-1 vaccine developments.

Optimizing HIV-1-specific CD8+ T-cell induction by recombinant BCG in prime-boost regimens with heterologous viral vectors

The desire to induce HIV‐1‐specific responses soon after birth to prevent breast milk transmission of HIV‐1 led us to propose a vaccine regimen which primes HIV‐1‐specific T cells using a recombinant Mycobacterium bovis bacillus Calmette‐Guérin (rBCG) vaccine. Because attenuated live bacterial vaccines are typically not sufficiently immunogenic as stand‐alone vaccines, rBCG‐primed T cells will likely require boost immunization(s). Here, we compared modified Danish (AERAS‐401) and Pasteur lysine auxotroph (222) strains of BCG expressing the immunogen HIVA for their potency to prime HIV‐1‐specific responses in adult BALB/c mice and examined four heterologous boosting HIVA vaccines for their immunogenic synergy. We found that both BCG.HIVA401 and BCG.HIVA222 primed HIV‐1‐specific CD8+ T‐cell‐mediated responses. The strongest boosts were delivered by human adenovirus‐vectored HAdV5.HIVA and sheep atadenovirus‐vectored OAdV7.HIVA vaccines, followed by poxvirus MVA.HIVA; the weakest was plasmid pTH.HIVA DNA. The prime‐boost regimens induced T cells capable of efficient in vivo killing of sensitized target cells. We also observed that the BCG.HIVA401 and BCG.HIVA222 vaccines have broadly similar immunologic properties, but display a number of differences mainly detected through distinct profiles of soluble intercellular signaling molecules produced by immune splenocytes in response to both HIV‐1‐ and BCG‐specific stimuli. These results encourage further development of the rBCG prime‐boost regimen.

Novel Recombinant Mycobacterium bovis BCG, Ovine Atadenovirus, and Modified Vaccinia Virus Ankara Vaccines Combine To Induce Robust Human Immunodeficiency Virus-Specific CD4 and CD8 T-Cell Responses in Rhesus Macaques

ABSTRACT Mycobacterium bovis bacillus Calmette-Guérin (BCG), which elicits a degree of protective immunity against tuberculosis, is the most widely used vaccine in the world. Due to its persistence and immunogenicity, BCG has been proposed as a vector for vaccines against other infections, including HIV-1. BCG has a very good safety record, although it can cause disseminated disease in immunocompromised individuals. Here, we constructed a recombinant BCG vector expressing HIV-1 clade A-derived immunogen HIVA using the recently described safer and more immunogenic BCG strain AERAS-401 as the parental mycobacterium. Using routine ex vivo T-cell assays, BCG.HIVA401 as a stand-alone vaccine induced undetectable and weak CD8 T-cell responses in BALB/c mice and rhesus macaques, respectively. However, when BCG.HIVA401 was used as a priming component in heterologous vaccination regimens together with recombinant modified vaccinia virus Ankara-vectored MVA.HIVA and ovine atadenovirus-vectored OAdV.HIVA vaccines, robust HIV-1-specific T-cell responses were elicited. These high-frequency T-cell responses were broadly directed and capable of proliferation in response to recall antigen. Furthermore, multiple antigen-specific T-cell clonotypes were efficiently recruited into the memory pool. These desirable features are thought to be associated with good control of HIV-1 infection. In addition, strong and persistent T-cell responses specific for the BCG-derived purified protein derivative (PPD) antigen were induced. This work is the first demonstration of immunogenicity for two novel vaccine vectors and the corresponding candidate HIV-1 vaccines BCG.HIVA401 and OAdV.HIVA in nonhuman primates. These results strongly support their further exploration.

Effects of protein molecular weight on the intrinsic material properties and release kinetics of wet spun polymeric microfiber delivery systems.

Wet spun microfibers have great potential for the design of multifunctional controlled release scaffolds. Understanding aspects of drug delivery and mechanical strength, specific to protein molecular weight, may aid in the optimization and development of wet spun fiber platforms. This study investigated the intrinsic material properties and release kinetics of poly(l-lactic acid) (PLLA) and poly(lactic-co-glycolic acid) (PLGA) wet spun microfibers encapsulating proteins with varying molecular weights. A cryogenic emulsion technique developed in our laboratory was used to encapsulate insulin (5.8 kDa), lysozyme (14.3 kDa) and bovine serum albumin (BSA, 66.0 kDa) within wet spun microfibers (~100 μm). Protein loading was found to significantly influence mechanical strength and drug release kinetics of PLGA and PLLA microfibers in a molecular-weight-dependent manner. BSA encapsulation resulted in the most significant decrease in strength and ductility for both PLGA and PLLA microfibers. Interestingly, BSA-loaded PLGA microfibers had a twofold increase (8±2 MPa to 16±1 MPa) in tensile strength and a fourfold increase (3±1% to 12±6%) in elongation until failure in comparison to PLLA microfibers. PLGA and PLLA microfibers exhibited prolonged protein release up to 63 days in vitro. Further analysis with the Korsmeyer-Peppas kinetic model determined that the mechanism of protein release was dependent on Fickian diffusion. These results emphasize the critical role protein molecular weight has on the properties of wet spun filaments, highlighting the importance of designing small molecular analogues to replace growth factors with large molecular weights.

Dual neonate vaccine platform against HIV-1 and M. tuberculosis.

Acquired immunodeficiency syndrome and tuberculosis (TB) are two of the worlds most devastating diseases. The first vaccine the majority of infants born in Africa receive is Mycobacterium bovis bacillus Calmette-Guérin (BCG) as a prevention against TB. BCG protects against disseminated disease in the first 10 years of life, but provides a variable protection against pulmonary TB and enhancing boost delivered by recombinant modified vaccinia virus Ankara (rMVA) expressing antigen 85A (Ag85A) of M. tuberculosis is currently in phase IIb evaluation in African neonates. If the newborns mother is positive for human immunodeficiency virus type 1 (HIV-1), the baby is at high risk of acquiring HIV-1 through breastfeeding. We suggested that a vaccination consisting of recombinant BCG expressing HIV-1 immunogen administered at birth followed by a boost with rMVA sharing the same immunogen could serve as a strategy for prevention of mother-to-child transmission of HIV-1 and rMVA expressing an African HIV-1-derived immunogen HIVA is currently in phase I trials in African neonates. Here, we aim to develop a dual neonate vaccine platform against HIV-1 and TB consisting of BCG.HIVA administered at birth followed by a boost with MVA.HIVA.85A. Thus, mMVA.HIVA.85A and sMVA.HIVA.85A vaccines were constructed, in which the transgene transcription is driven by either modified H5 or short synthetic promoters, respectively, and tested for immunogenicity alone and in combination with BCG.HIVA222. mMVA.HIVA.85A was produced markerless and thus suitable for clinical manufacture. While sMVA.HIVA.85A expressed higher levels of the immunogens, it was less immunogenic than mMVA.HIVA.85A in BALB/c mice. A BCG.HIVA222–mMVA.HIVA.85A prime-boost regimen induced robust T cell responses to both HIV-1 and M. tuberculosis. Therefore, proof-of-principle for a dual anti-HIV-1/M. tuberculosis infant vaccine platform is established. Induction of immune responses against these pathogens soon after birth is highly desirable and may provide a basis for lifetime protection maintained by boosts later in life.

Multifunctional polymeric microfibers with prolonged drug delivery and structural support capabilities

The strength and stability of hybrid fiber delivery systems, ones that perform a mechanical function and simultaneously deliver drug, are critical in the design of surgically implantable constructs. We report the fabrication of drug-eluting microfibers where drug loading and processing conditions alone increase microfiber strength and stability partially due to solvent-induced crystallization. Poly(L-lactic acid) microfibers of 64±7 μm diameter were wet spun by phase inversion. Encapsulation of a model hydrophobic anti-inflammatory drug, dexamethasone, at high loading provided stability to microfibers which maintained linear cumulative release kinetics up to 8 weeks in vitro. In our wet spinning process, all microfibers had increased crystallinity (13-17%) in comparison to unprocessed polymer without any mechanical stretching. Moreover, microfibers with the highest drug loading retained 97% of initial tensile strength and were statistically stronger than all other microfiber formulations, including control fibers without drug. Results indicate that the encapsulation of small hydrophobic molecules (<400 Da) may increase the mechanical integrity of microfilaments whose crystallinity is also increased as a result of the process. Multifunctional drug-eluting microfibers can provide an exciting new opportunity to design novel biomaterials with mechanical stability and controlled release of a variety of therapeutics with micron-scale accuracy.

Bioengineered human and allogeneic pulmonary valve conduits chronically implanted orthotopically in baboons: Hemodynamic performance and immunologic consequences

OBJECTIVE This study assesses in a baboon model the hemodynamics and human leukocyte antigen immunogenicity of chronically implanted bioengineered (decellularized with collagen conditioning treatments) human and baboon heart valve scaffolds. METHODS Fourteen baboons underwent pulmonary valve replacement, 8 with decellularized and conditioned (bioengineered) pulmonary valves derived from allogeneic (N = 3) or xenogeneic (human) (N = 5) hearts; for comparison, 6 baboons received clinically relevant reference cryopreserved or porcine valved conduits. Panel-reactive serum antibodies (human leukocyte antigen class I and II), complement fixing antibodies (C1q binding), and C-reactive protein titers were measured serially until elective sacrifice at 10 or 26 weeks. Serial transesophageal echocardiograms measured valve function and geometry. Differences were analyzed with Kruskal-Wallis and Wilcoxon rank-sum tests. RESULTS All animals survived and thrived, exhibiting excellent immediate implanted valve function by transesophageal echocardiograms. Over time, reference valves developed a smaller effective orifice area index (median, 0.84 cm(2)/m(2); range, 1.22 cm(2)/m(2)), whereas all bioengineered valves remained normal (effective orifice area index median, 2.45 cm(2)/m(2); range, 1.35 cm(2)/m(2); P = .005). None of the bioengineered valves developed elevated peak transvalvular gradients: 5.5 (6.0) mm Hg versus 12.5 (23.0) mm Hg (P = .003). Cryopreserved valves provoked the most intense antibody responses. Two of 5 human bioengineered and 2 of 3 baboon bioengineered valves did not provoke any class I antibodies. Bioengineered human (but not baboon) scaffolds provoked class II antibodies. C1q(+) antibodies developed in 4 recipients. CONCLUSIONS Valve dysfunction correlated with markers for more intense inflammatory provocation. The tested bioengineering methods reduced antigenicity of both human and baboon valves. Bioengineered replacement valves from both species were hemodynamically equivalent to native valves.