Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage

The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus-based method. We first showed that poly-l-lysine could immobilize lentivirus to poly(ε-caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor β3 (TGF-β3), using a 3D woven poly(ε-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-β3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.The development of tissue constructs with matrix composition and biomechanical properties suitable for tissue replacement remains a significant goal of musculoskeletal tissue engineering. The canonical paradigm for engineering biological tissue substitutes generally involves three critical components (1, 2): (i) cells capable of populating the tissue substitute and generating appropriate neotissue constituents (3); (ii) biomimetic scaffolds that cells can infiltrate and that can support extracellular matrix (ECM) accumulation and organization (4); and (iii) environmental signals such as growth factors (5), small molecules, or physical factors (6) that can direct cell differentiation toward the appropriate lineage of choice. In traditional tissue engineering approaches, these elements are generally applied ex vivo to induce tissue-specific lineage commitment and matrix formation. Although promising strategies for developing bone, cartilage, tendon, and muscle tissue substitutes based on this paradigm exist (7–9), these approaches require extensive in vitro manipulation of engineered replacements. Furthermore, upon implantation in vivo, the ability to further specify or modify cell fate is lost.The ability to induce stem cell differentiation in situ in the absence of exogenous growth factors could significantly enhance tissue regeneration efforts by eliminating the need for in vitro culture of tissue replacements. Patient-derived cells could be harvested and seeded onto scaffolds in a single surgery, or progenitor cells may populate the scaffold in vivo following implantation. This approach requires a biomaterial that can provide functional tissue properties while neotissue is formed in response to persistent cues that guide in situ ECM development. In this regard, previous approaches have developed engineered constructs that elute small molecules or locally deliver proteins capable of initiating cell differentiation (10) or migration (11). Furthermore, efforts to enhance cell differentiation have involved functionalization of synthetic scaffolds with ECM proteins or peptides recognized by cell surface receptors (12–14). More recently, techniques for incorporating gene vectors, such as naked plasmid DNA or nonintegrating adenoviruses and adeno-associated viruses, into tissue replacement constructs have been used (15–19). These efforts rely on the ability of transient signals, limited by protein half-life or loss of transgene expression from nonintegrating vectors, to direct cell lineage commitment and drive ECM deposition. Moreover, whereas many of the substrates used in these studies enable localized gene delivery, the mechanical properties do not recapitulate those of native tissues. Thus, implantation of such constructs must be delayed until after ex vivo development of neotissue to allow the implant to function in the context of normal physiological loads.The goal of this study was to develop a bioactive scaffold capable of mediating cell differentiation and formation of an ECM suitable for replacement of musculoskeletal tissues with mechanical properties that mimic those of native tissues. Lentiviral vectors were used due to their capacity to efficiently transduce target cells and induce sustained expression of transgenes relevant to cell differentiation and ECM formation in the context of cartilage tissue engineering. Moreover, as poly-l-lysine (PLL)-coated biomaterials effectively immobilize other enveloped viruses and facilitate efficient transduction of cells (20), PLL was used to functionalize poly(ε-caprolactone) (PCL)-derived biomaterials with lentivirus (LV). In this study, we first tested whether PLL could effectively immobilize LV to PCL films and lead to efficient transduction of human mesenchymal stem cells (hMSCs). We then tested whether scaffold-mediated gene delivery with a biomechanically relevant, 3D orthogonally woven PCL scaffold could influence ECM formation by hMSCs in chondrogenic cultures.     

Absence of posttraumatic arthritis following intraarticular fracture in the MRL/MpJ mouse

OBJECTIVE: Posttraumatic arthritis is a frequent long-term complication of intraarticular fractures. A model of a closed intraarticular fracture in C57BL/6 mice that progresses to posttraumatic arthritis has been developed. The MRL/MpJ mouse has shown unique regenerative abilities in response to injury. The objective of this study was to determine if the MRL/MpJ mouse is protected from posttraumatic arthritis after intraarticular fractures. METHODS: Intraarticular fractures were created in MRL/MpJ mice and C57BL/6 control mice (n = 16 each). Limbs were analyzed for posttraumatic arthritis 4 and 8 weeks after fracture using microfocal computed tomography bone morphology, subchondral bone thickness evaluation, and histologic evaluation of cartilage degeneration. Serum cytokines and biomarkers were measured after the mice were killed. RESULTS: Intraarticular fractures were successfully created in all 32 mice. In the experimental fractured limbs, C57BL/6 mice had a decrease in bone density, increased subchondral bone thickness, and increased cartilage degeneration compared with normal contralateral control limbs. In the MRL/MpJ mice, no differences in bone density, subchondral bone thickness, or histologic grading of cartilage degeneration were seen between fractured and contralateral control limbs. Cytokine analysis showed lower systemic levels of the proinflammatory cytokine interleukin-1alpha (IL-1alpha) and higher levels of the antiinflammatory cytokines IL-4 and IL-10 in the MRL/MpJ mice. CONCLUSION: This study shows that the MRL/MpJ mouse is relatively protected from posttraumatic arthritis after intraarticular fracture. Further investigation into the mechanism involved in this response will hopefully provide new insight into the pathogenesis, prevention, and treatment of posttraumatic arthritis after intraarticular fracture.     

Long-term comparison of the outcomes of Ahmed Glaucoma Valve surgery between glaucoma surgeons and cornea trained surgeons

International Ophthalmology - To compare the long-term outcomes obtained by glaucoma surgeons versus cornea trained surgeons performing Ahmed glaucoma valve (AGV) surgery. Of the total 376 patients...     

Outcome of therapy‐related acute promyelocytic leukemia with or without arsenic trioxide as a component of frontline therapy

BACKGROUND:

Patients with therapy‐related acute promyelocytic leukemia (t‐APL) have been commonly exposed to topoisomerase inhibitors and may potentially benefit from induction regimens omitting anthracyclines.

METHODS:

Retrospective analysis of the outcomes of 29 patients with t‐APL who were either treated with arsenic trioxide (ATO) and all‐trans‐retinoic acid (ATRA) or with standard ATRA plus anthracycline‐based chemotherapy was performed.

RESULTS:

Prior therapy included chemotherapy alone, radiation alone, or a combination of the 2 in 19%, 33%, and 47% of patients, respectively. The combination of ATO and ATRA (n = 19) for induction resulted in a similar remission rate compared with ATRA plus chemotherapy (n = 10) (89% vs 70%; P = .35). The median overall survival for the patients treated with ATRA plus ATO was not reached compared with that for patients treated with ATRA plus chemotherapy (161 weeks; P = .79).

CONCLUSIONS:

In this cohort of t‐APL patients, outcomes with ATO and ATRA appeared to be comparable to anthracycline‐containing induction regimens. This combination may be preferable in t‐APL patients to avoid any risk of anthracycline‐induced toxicities. Cancer 2011. © 2010 American Cancer Society.     

Internal urethrotomy and intraurethral submucosal injection of triamcinolone in short bulbar urethral strictures

Objectives

In clinical practice, internal urethrotomy is an easy procedure and is offered as a first modality for treatment of short urethral strictures. Internal urethrotomy refers to any procedure that opens the stricture by incising or ablating it transurethrally. The most common complication of internal urethrotomy is stricture recurrence. The curative success rate of internal urethrotomy is approximately 20%. Triamcinolone has antifibroblast and anticollagen properties. This study evaluated the efficacy of triamcinolone in the prevention of anterior urethral stricture recurrence after internal urethrotomy.

Methods

Fifty male patients with anterior urethral stricture were randomized to undergo internal urethrotomy with or without urethral submucosal injection of triamcinolone. Using general anesthesia urethrotomy was performed. Triamcinolone (40 mg) was injected submucosally at the urethrotomy site in 25 patients. The patients were followed for at least 12 months and the stricture recurrence rate was compared between the two groups.

Results

23 patients in the triamcinolone group and 22 in the control group completed the study. There were no significant differences in the baseline characteristics of the patients or the etiology of the stricture between the two groups. Mean follow-up time was 13.7 ± 5.5 months (range: 1–25 months). Urethral stricture recurred in five patients (21.7%) in the triamcinolone group and in 11 patients (50%) in the control group (P = 0.04).

Conclusions

Injection of triamcinolone significantly reduced stricture recurrence after internal urethrotomy. Further investigations are warranted to confirm its efficacy and safety.     

Adipose-derived stromal vascular fraction improves tendon healing in rabbits

Objective:To evaluate the potential effects of uncultured adipose-derived stromal vascular fraction on tendon healing.Methods:Twenty five adult male New Zealand white rabbits weighing 2.5-3.0 kg were used.Five rabbits were used as donors of adipose tissue and the rest were divided into control and treatment groups.The injury model was completed by unilateral tenotomy through the middle one third of deep digital flexor tendon.Immediately after suture repair,either fresh stromal vascular fraction from enzymatic digestion of adipose tissue or placebo was intratendinously injected at tendon stumps in treatment and control groups,respectively.Immobilization with cast was continued for two weeks after surgery.Animals were sacrificed at eight weeks after surgery and tendons underwent histological,immunohistochemical,and mechanical evaluations.Statistical analyses of quantitative and qualitative data were assessed using one-way analysis of variance and MannWhitney U-test,respectively.Results:Histological evaluations demonstrated superior fibrillar linearity and continuity,and decreased vascularity in treatment group indicated improved organization and remodeling of neotendons.Immunohistochemistry demonstrated a significant increase in collagen I expression in treatment group.Ultimate load and energy absorption capacity were both significantly increased in cell-treated repairs compared with controls.Conclusion: The present study shows that intratendinous injection of uncultured adipose-derived stromal vascular fraction results in improved structural and mechanical properties of tendon repairs and it could be an effective modality for treating tendon injury.     

Phase 2 clinical trial of a recombinant adeno-associated viral vector expressing α1-antitrypsin: interim results

Recombinant adeno-associated virus (rAAV) vectors offer promise for the gene therapy of α(1)-antitrypsin (AAT) deficiency. In our prior trial, an rAAV vector expressing human AAT (rAAV1-CB-hAAT) provided sustained, vector-derived AAT expression for >1 year. In the current phase 2 clinical trial, this same vector, produced by a herpes simplex virus complementation method, was administered to nine AAT-deficient individuals by intramuscular injection at doses of 6.0×10(11), 1.9×10(12), and 6.0×10(12) vector genomes/kg (n=3 subjects/dose). Vector-derived expression of normal (M-type) AAT in serum was dose dependent, peaked on day 30, and persisted for at least 90 days. Vector administration was well tolerated, with only mild injection site reactions and no serious adverse events. Serum creatine kinase was transiently elevated on day 30 in five of six subjects in the two higher dose groups and normalized by day 45. As expected, all subjects developed anti-AAV antibodies and interferon-γ enzyme-linked immunospot responses to AAV peptides, and no subjects developed antibodies to AAT. One subject in the mid-dose group developed T cell responses to a single AAT peptide unassociated with any clinical effects. Muscle biopsies obtained on day 90 showed strong immunostaining for AAT and moderate to marked inflammatory cell infiltrates composed primarily of CD3-reactive T lymphocytes that were primarily of the CD8(+) subtype. These results support the feasibility and safety of AAV gene therapy for AAT deficiency, and indicate that serum levels of vector-derived normal human AAT >20?μg/ml can be achieved. However, further improvements in the design or delivery of rAAV-AAT vectors will be required to achieve therapeutic target serum AAT concentrations.     

Chondroprotective effects of a polycarbonate-urethane meniscal implant: histopathological results in a sheep model

Purpose

Injury or loss of the meniscus generally leads to degenerative osteoarthritic changes in the knee joint. However, few surgical options exist for meniscal replacement. The goal of this study was to examine the ability of a non-degradable, anatomically shaped artificial meniscal implant, composed of Kevlar^?-reinforced polycarbonate-urethane (PCU), to prevent progressive cartilage degeneration following complete meniscectomy.

Methods

The artificial meniscus was implanted in the knees of mature female sheep following total medial meniscectomy, and the animals were killed at 3- and 6-months post-surgery. Macroscopic analysis and semi-quantitative histological analysis were performed on the cartilage of the operated knee and unoperated contralateral control joint.

Results

The PCU implants remained well secured throughout the experimental period and showed no signs of wear or changes in structural or material properties. Histological analysis showed relatively mild cartilage degeneration that was dominated by loss of proteoglycan content and cartilage structure. However, the total osteoarthritis score did not significantly differ between the control and operated knees, and there were no differences in the severity of degenerative changes between 3 and 6?months post-surgery.

Conclusion

Current findings provide preliminary evidence for the ability of an artificial PCU meniscal implant to delay or prevent osteoarthritic changes in knee joint following complete medial meniscectomy.