Optimized Media Volumes Enable Homogeneous Growth of Mesenchymal Stem Cell-Based Engineered Cartilage Constructs.

Despite marked advances in the field of cartilage tissue engineering, it remains a challenge to engineer cartilage constructs with homogeneous properties. Moreover, for engineered cartilage to make it to the clinic, this homogeneous growth must occur in a time-efficient manner. In this study we investigated the potential of increased media volume to expedite the homogeneous maturation of mesenchymal stem cell (MSC) laden engineered constructs over time in vitro. We assessed the MSC-laden constructs after 4 and 8 weeks of chondrogenic culture using bulk mechanical, histological, and biochemical measures. These assays were performed on both the intact total constructs and the construct cores to elucidate region-dependent differences. In addition, local strain transfer was assessed to quantify depth-dependent mechanical properties throughout the constructs. Our findings suggest that increased media volume enhances matrix deposition early in culture and ameliorates unwanted regional heterogeneities at later time points. Taken together, these data support the use of higher media volumes during in vitro culture to hasten tissue maturation and increase the core strength of tissue constructs. These findings will forward the field of cartilage tissue engineering and the translation of tissue engineered constructs.

Ethics of Using Animal Models as Predictors of Human Response in Tissue Engineering.

Use of outcomes from animal research for prediction of human response in tissue engineering studies has many ethical considerations. This article aims to contribute to the ethical discussion by delineating the framework of animal research and the ethical considerations at play, in particular with respect to cartilage tissue engineering. The history of animal research regulation and the current status of animal research in orthopedic tissue engineering is discussed. Questions addressed include how the proper animal models are chosen, how regulatory bodies ensure animal wellness and safety, and how guidelines are implemented and maintained throughout the life cycle of a project. Finally, we provide examples of both in vitro and in vivo cartilage tissue engineering research where animal models were employed as a predictive model of human response.

Age-Dependent Subchondral Bone Remodeling and Cartilage Repair in a Minipig Defect Model.

After cartilage injury and repair, the subchondral bone plate remodels. Skeletal maturity likely impacts both bone remodeling and inherent cartilage repair capacity. The objective of this study was to evaluate subchondral bone remodeling as a function of injury type, repair scenario, and skeletal maturity in a Yucatan minipig model. Cartilage defects (4 mm) were created bilaterally in the trochlear groove. Treatment conditions included a full thickness chondral defect (full chondral defect, n = 3 adult/3 juvenile), a partial thickness (∼50%) chondral defect (PCD, n = 3/3), and FCD treated with microfracture (MFX, n = 3/3). At 6 weeks postoperatively, osteochondral samples containing the lesion site were imaged by micro-computed tomography (CT) and analyzed by histology and immunohistochemistry. Via micro-CT, FCD and MFX groups showed increased bone loss in juveniles compared with adults. Quantification of histology using the ICRS II scoring system showed equal overall assessment for the FCD groups and better overall assessment in juvenile animals treated with MFX compared with adults. All FCD and MFX groups were inferior to control samples. For the PCD injury, both age groups had values close to the control values. For the FCD groups, there were greater alterations in the subchondral bone in juveniles compared with adults. Staining for collagen II showed more intense signals in juvenile FCD and MFX groups compared with adults. This large animal study of cartilage repair shows the significant impact of skeletal maturity on the propensity of subchondral bone to remodel as a result of chondral injury. This will improve selection criteria for animal models for studying cartilage injury, repair, and treatment.

* Thermosensitive Poly(N-vinylcaprolactam) Injectable Hydrogels for Cartilage Tissue Engineering.

Injectable hydrogels have gained prominence in the field of tissue engineering for minimally invasive delivery of cells for tissue repair and in the filling of irregular defects. However, many injectable hydrogels exhibit long gelation times or are not stable for long periods after injection. To address these concerns, we used thermosensitive poly(N-vinylcaprolactam) (PNVCL) hydrogels due to their cytocompatibility and fast response to temperature stimuli. Changes in the PNVCL molecular weight and concentration enabled the development of hydrogels with tunable mechanical properties and fast gelation times (<60 s when the temperature was raised from room temperature to physiologic temperature). Chondrocytes (CHs) and mesenchymal stem cells were encapsulated in PNVCL hydrogels and exhibited high viability (∼90%), as monitored by Live/Dead staining and Alamar Blue assays. Three-dimensional constructs of CH-laden PNVCL hydrogels supported cartilage-specific extracellular matrix production both in vitro and after subcutaneous injection in nude rats for up to 8 weeks. Moreover, biochemical analyses of constructs demonstrated a time-dependent increase in glycosaminoglycans (GAGs) and collagen, which were significantly augmented in the implants cultured in vivo. Histological analyses also demonstrated regular distribution of synthesized cartilage components, including abundant GAGs and type II collagen. The findings from this study demonstrate thermosensitive PNVCL as a candidate injectable biomaterial to deliver cells for cartilage tissue engineering.

Effects of Mesenchymal Stem Cell and Growth Factor Delivery on Cartilage Repair in a Mini-Pig Model.

OBJECTIVE: We have recently shown that mesenchymal stem cells (MSCs) embedded in a hyaluronic acid (HA) hydrogel and exposed to chondrogenic factors (transforming growth factor-ß3 [TGF-ß3]) produce a cartilage-like tissue in vitro. The current objective was to determine if these same factors could be combined immediately prior to implantation to induce a superior healing response in vivo relative to the hydrogel alone. DESIGN: Trochlear chondral defects were created in Yucatan mini-pigs (6 months old). Treatment groups included an HA hydrogel alone and hydrogels containing allogeneic MSCs, TGF-ß3, or both. Six weeks after surgery, micro-computed tomography was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. RESULTS: Treatment with TGF-ß3 led to a marked increase in positive staining for collagen type II within defects (P < 0.05), while delivery of MSCs did not (P > 0.05). Neither condition had an impact on other histological semiquantitative scores (P > 0.05), and inclusion of MSCs led to significantly less defect fill (P < 0.05). For all measurements, no synergistic interaction was found between TGF-ß3 and MSC treatment when they were delivered together (P > 0.05). CONCLUSIONS: At this early healing time point, treatment with TGF-ß3 promoted the formation of collagen type II within the defect, while allogeneic MSCs had little benefit. Combination of TGF-ß3 and MSCs at the time of surgery did not produce a synergistic effect. An in vitro precultured construct made of these components may be required to enhance in vivo repair in this model system.

Engineering meniscus structure and function via multi-layered mesenchymal stem cell-seeded nanofibrous scaffolds.

Despite advances in tissue engineering for the knee meniscus, it remains a challenge to match the complex macroscopic and microscopic structural features of native tissue, including the circumferentially and radially aligned collagen bundles essential for mechanical function. To mimic this structural hierarchy, this study developed multi-lamellar mesenchymal stem cell (MSC)-seeded nanofibrous constructs. Bovine MSCs were seeded onto nanofibrous scaffolds comprised of poly(ε-caprolactone) with fibers aligned in a single direction (0° or 90° to the scaffold long axis) or circumferentially aligned (C). Multi-layer groups (0°/0°/0°, 90°/90°/90°, 0°/90°/0°, 90°/0°/90°, and C/C/C) were created and cultured for a total of 6 weeks under conditions favoring fibrocartilaginous tissue formation. Tensile testing showed that 0° and C single layer constructs had stiffness values several fold higher than 90° constructs. For multi-layer groups, the stiffness of 0°/0°/0° constructs was higher than all other groups, while 90°/90°/90° constructs had the lowest values. Data for collagen content showed a general positive interactive effect for multi-layers relative to single layer constructs, while a positive interaction for stiffness was found only for the C/C/C group. Collagen content and cell infiltration occurred independent of scaffold alignment, and newly formed collagenous matrix followed the scaffold fiber direction. Structural hierarchies within multi-lamellar constructs dictated biomechanical properties, and only the C/C/C constructs with non-orthogonal alignment within layers featured positive mechanical reinforcement as a consequence of the layered construction. These multi-layer constructs may serve as functional substitutes for the meniscus as well as test beds to understand the complex mechanical principles that enable meniscus function.

Cartilage repair and subchondral bone remodeling in response to focal lesions in a mini-pig model: implications for tissue engineering.

OBJECTIVE: Preclinical large animal models are essential for evaluating new tissue engineering (TE) technologies and refining surgical approaches for cartilage repair. Some preclinical animal studies, including the commonly used minipig model, have noted marked remodeling of the subchondral bone. However, the mechanisms underlying this response have not been well characterized. Thus, our objective was to compare in-vivo outcomes of chondral defects with varied injury depths and treatments. DESIGN: Trochlear chondral defects were created in 11 Yucatan minipigs (6 months old). Groups included an untreated partial-thickness defect (PTD), an untreated full-thickness defect (FTD), and FTDs treated with microfracture, autologous cartilage transfer (FTD-ACT), or an acellular hyaluronic acid hydrogel. Six weeks after surgery, micro-computed tomography (µCT) was used to quantitatively assess defect fill and subchondral bone remodeling. The quality of cartilage repair was assessed using the ICRS-II histological scoring system and immunohistochemistry for type II collagen. A finite element model (FEM) was developed to assess load transmission. RESULTS: Using µCT, substantial bone remodeling was observed for all FTDs, but not for the PTD group. The best overall histological scores and greatest type II collagen staining was found for the FTD-ACT and PTD groups. The FEM confirmed that only the FTD-ACT group could initially restore appropriate transfer of compressive loads to the underlying bone. CONCLUSIONS: The bony remodeling observed in this model system appears to be a biological phenomena and not a result of altered mechanical loading, with the depth of the focal chondral defect (partial vs. full thickness) dictating the bony remodeling response. The type of cartilage injury should be carefully controlled in studies utilizing this model to evaluate TE approaches for cartilage repair.

Repair of dense connective tissues via biomaterial-mediated matrix reprogramming of the wound interface.

Repair of dense connective tissues in adults is limited by their intrinsic hypocellularity and is exacerbated by a dense extracellular matrix (ECM) that impedes cellular migration to and local proliferation at the wound site. Conversely, healing in fetal tissues occurs due in part to an environment conducive to cell mobility and division. Here, we investigated whether the application of a degradative enzyme, collagenase, could reprogram the adult wound margin to a more fetal-like state, and thus abrogate the biophysical impediments that hinder migration and proliferation. We tested this concept using the knee meniscus, a commonly injured structure for which few regenerative approaches exist. To focus delivery and degradation to the wound interface, we developed a system in which collagenase was stored inside poly(ethylene oxide) (PEO) electrospun nanofibers and released upon hydration. Through a series of in vitro and in vivo studies, our findings show that partial digestion of the wound interface improves repair by creating a more compliant and porous microenvironment that expedites cell migration to and/or proliferation at the wound margin. This innovative approach of targeted manipulation of the wound interface, focused on removing the naturally occurring barriers to adult tissue repair, may find widespread application in the treatment of injuries to a variety of dense connective tissues.

Maximizing cartilage formation and integration via a trajectory-based tissue engineering approach.

Given the limitations of current surgical approaches to treat articular cartilage injuries, tissue engineering (TE) approaches have been aggressively pursued. Despite reproduction of key mechanical attributes of native tissue, the ability of TE cartilage constructs to integrate with native tissue must also be optimized for clinical success. In this paper, we propose a "trajectory-based" tissue engineering (TB-TE) approach, based on the hypothesis that time-dependent increases in construct maturation in-vitro prior to implantation (i.e. positive rates) may provide a reliable predictor of in-vivo success. As an example TE system, we utilized hyaluronic acid hydrogels laden with mesenchymal stem cells. We first modeled the maturation of these constructs in-vitro to capture time-dependent changes. We then performed a sensitivity analysis of the model to optimize the timing and amount of data collection. Finally, we showed that integration to cartilage in-vitro is not correlated to the maturation state of TE constructs, but rather their maturation rate, providing a proof-of-concept for the use of TB-TE to enhance treatment outcomes following cartilage injury. This new approach challenges the traditional TE paradigm of matching only native state parameters of maturity and emphasizes the importance of also establishing an in-vitro trajectory in constructs in order to improve the chance of in-vivo success.

Organized nanofibrous scaffolds that mimic the macroscopic and microscopic architecture of the knee meniscus.

The menisci are crescent-shaped fibrocartilaginous tissues whose structural organization consists of dense collagen bundles that are locally aligned but show a continuous change in macroscopic directionality. This circumferential patterning is necessary for load transmission across the knee joint and is a key design parameter for tissue engineered constructs. To address this issue we developed a novel electrospinning method to produce scaffolds composed of circumferentially aligned (CircAl) nanofibers, quantified their structure and mechanics, and compared them with traditional linearly aligned (LinAl) scaffolds. Fibers were locally oriented in CircAl scaffolds, but their orientation varied considerably as a function of position (P<0.05). LinAl fibers did not change in orientation over a similar length scale (P>0.05). Cell seeding of CircAl scaffolds resulted in a similar cellular directionality. Mechanical analysis of CircAl scaffolds revealed significant interactions between scaffold length and region (P<0.05), with the tensile modulus near the edge of the scaffolds decreasing with increasing scaffold length. No such differences were detected in LinAl specimens (P>0.05). Simulation of the fiber deposition process produced "theoretical" fiber populations that matched the fiber organization and mechanical properties observed experimentally. These novel scaffolds, with spatially varying local orientations and mechanics, will enable the formation of functional anatomic meniscus constructs.

Primary infection by a human immunodeficiency virus with atypical coreceptor tropism.

The great majority of human immunodeficiency virus type 1 (HIV-1) strains enter CD4+ target cells by interacting with one of two coreceptors, CCR5 or CXCR4. Here we describe a transmitted/founder (T/F) virus (ZP6248) that was profoundly impaired in its ability to utilize CCR5 and CXCR4 coreceptors on multiple CD4+ cell lines as well as primary human CD4+ T cells and macrophages in vitro yet replicated to very high titers (>80 million RNA copies/ml) in an acutely infected individual. Interestingly, the envelope (Env) glycoprotein of this clade B virus had a rare GPEK sequence in the crown of its third variable loop (V3) rather than the consensus GPGR sequence. Extensive sequencing of sequential plasma samples showed that the GPEK sequence was present in virtually all Envs, including those from the earliest time points after infection. The molecularly cloned (single) T/F virus was able to replicate, albeit poorly, in cells obtained from ccr5Δ32 homozygous donors. The ZP6248 T/F virus could also infect cell lines overexpressing the alternative coreceptors GPR15, APJ, and FPRL-1. A single mutation in the V3 crown sequence (GPEK->GPGK) of ZP6248 restored its infectivity in CCR5+ cells but reduced its ability to replicate in GPR15+ cells, indicating that the V3 crown motif played an important role in usage of this alternative coreceptor. These results suggest that the ZP6248 T/F virus established an acute in vivo infection by using coreceptor(s) other than CCR5 or CXCR4 or that the CCR5 coreceptor existed in an unusual conformation in this individual.

Phenotypic and immunologic comparison of clade B transmitted/founder and chronic HIV-1 envelope glycoproteins.

Sexual transmission of human immunodeficiency virus type 1 (HIV-1) across mucosal barriers is responsible for the vast majority of new infections. This relatively inefficient process results in the transmission of a single transmitted/founder (T/F) virus, from a diverse viral swarm in the donor, in approximately 80% of cases. Here we compared the biological activities of 24 clade B T/F envelopes (Envs) with those from 17 chronic controls to determine whether the genetic bottleneck that occurs during transmission is linked to a particular Env phenotype. To maximize the likelihood of an intact mucosal barrier in the recipients and to enhance the sensitivity of detecting phenotypic differences, only T/F Envs from individuals infected with a single T/F variant were selected. Using pseudotyping to assess Env function in single-round infectivity assays, we compared coreceptor tropism, CCR5 utilization efficiencies, primary CD4(+) T cell subset tropism, dendritic cell trans-infections, fusion kinetics, and neutralization sensitivities. T/F and chronic Envs were phenotypically equivalent in most assays; however, T/F Envs were modestly more sensitive to CD4 binding site antibodies b12 and VRC01, as well as pooled human HIV Ig. This finding was independently validated with a panel of 14 additional chronic HIV-1 Env controls. Moreover, the enhanced neutralization sensitivity was associated with more efficient binding of b12 and VRC01 to T/F Env trimers. These data suggest that there are subtle but significant structural differences between T/F and chronic clade B Envs that may have implications for HIV-1 transmission and the design of effective vaccines.

A maraviroc-resistant HIV-1 with narrow cross-resistance to other CCR5 antagonists depends on both N-terminal and extracellular loop domains of drug-bound CCR5.

CCR5 antagonists inhibit HIV entry by binding to a coreceptor and inducing changes in the extracellular loops (ECLs) of CCR5. In this study, we analyzed viruses from 11 treatment-experienced patients who experienced virologic failure on treatment regimens containing the CCR5 antagonist maraviroc (MVC). Viruses from one patient developed high-level resistance to MVC during the course of treatment. Although resistance to one CCR5 antagonist is often associated with broad cross-resistance to other agents, these viruses remained sensitive to most other CCR5 antagonists, including vicriviroc and aplaviroc. MVC resistance was dependent upon mutations within the V3 loop of the viral envelope (Env) protein and was modulated by additional mutations in the V4 loop. Deep sequencing of pretreatment plasma viral RNA indicated that resistance appears to have occurred by evolution of drug-bound CCR5 use, despite the presence of viral sequences predictive of CXCR4 use. Envs obtained from this patient before and during MVC treatment were able to infect cells expressing very low CCR5 levels, indicating highly efficient use of a coreceptor. In contrast to previous reports in which CCR5 antagonist-resistant viruses interact predominantly with the N terminus of CCR5, these MVC-resistant Envs were also dependent upon the drug-modified ECLs of CCR5 for entry. Our results suggest a model of CCR5 cross-resistance whereby viruses that predominantly utilize the N terminus are broadly cross-resistant to multiple CCR5 antagonists, whereas viruses that require both the N terminus and antagonist-specific ECL changes demonstrate a narrow cross-resistance profile.