Allogeneic Versus Autologous Derived Cell Sources for Use in Engineered Bone-Ligament-Bone Grafts in Sheep Anterior Cruciate Ligament Repair

The use of autografts versus allografts for anterior cruciate ligament (ACL) reconstruction is controversial. The current popular options for ACL reconstruction are patellar tendon or hamstring autografts, yet advances in allograft technologies have made allogeneic grafts a favorable option for repair tissue. Despite this, the mismatched biomechanical properties and risk of osteoarthritis resulting from the current graft technologies have prompted the investigation of new tissue sources for ACL reconstruction. Previous work by our lab has demonstrated that tissue-engineered bone-ligament-bone (BLB) constructs generated from an allogeneic cell source develop structural and functional properties similar to those of native ACL and vascular and neural structures that exceed those of autologous patellar tendon grafts. In this study, we investigated the effectiveness of our tissue-engineered ligament constructs fabricated from autologous versus allogeneic cell sources. Our preliminary results demonstrate that 6 months postimplantation, our tissue-engineered auto- and allogeneic BLB grafts show similar histological and mechanical outcomes indicating that the autologous grafts are a viable option for ACL reconstruction. These data indicate that our tissue-engineered autologous ligament graft could be used in clinical situations where immune rejection and disease transmission may preclude allograft use.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140234/1/ten.tea.2014.0422.pd

Fresh Versus Frozen Engineered Bone–Ligament–Bone Grafts for Sheep Anterior Cruciate Ligament Repair

Surgical intervention is often required to restore knee instability in patients with anterior cruciate ligament (ACL) injury. The most commonly used grafts for ACL reconstruction are tendon autografts or allografts. These current options, however, have shown failure rates requiring revision and continued instability in the long term. The mismatched biomechanical properties of the current tendon grafts compared with native ACL tissue are thought to contribute to these poor outcomes and potential risk of early onset osteoarthritis. As a possible solution to these issues, our laboratory has fabricated tissue-engineered ligament constructs that exhibit structural and functional properties similar to those of native ACL tissue after 6 months implantation. In addition, these tissue-engineered grafts achieve vascular and neural development that exceeds those of patellar tendon grafts. However, the utility of our tissue-engineered grafts is limited by the labor-intensive method required to produce the constructs and the need to use the constructs fresh, directly from the cell culturing system. Ideally, these constructs would be fabricated and stored until needed. Thus, in this study, we investigated the efficacy of freezing our tissue-engineered constructs as a method of preservation before use for ACL reconstruction. We hypothesized that frozen constructs would have similar histological and biomechanical outcomes compared with our fresh model. Our results showed that 6 months postimplantation as an ACL replacement graft, both our tissue-engineered fresh and frozen grafts demonstrated similar mechanical and histological outcomes, indicating that freezing is a suitable method for preserving and storing our graft before ACL reconstruction. The ability to use frozen constructs significantly increases the versatility of our graft technology expanding the clinical utility of our graft.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/140250/1/ten.tec.2014.0542.pd

Localization, Shedding, Regulation and Function of Aminopeptidase N/CD13 on Fibroblast like Synoviocytes.

Aminopeptidase N/CD13 is highly expressed by fibroblast like synoviocytes (FLS) and may play a role in rheumatoid arthritis (RA). CD13 was previously detected in human synovial fluid where it was significantly increased in RA compared to osteoarthritis. In this study we found that CD13 in biological fluids (plasma, synovial fluid, FLS culture supernatant) is present as both a soluble molecule and on extracellular vesicles, including exosomes, as assessed by differential ultracentrifugation and density gradient separation. Having determined CD13 could be released as a soluble molecule from FLS, we examined potential mechanisms by which CD13 might be shed from the FLS membrane. The use of protease inhibitors revealed that CD13 is cleaved from the FLS surface by metalloproteinases. siRNA treatment of FLS revealed one of those proteases to be MMP14. We determined that pro-inflammatory cytokines (TNFα, IFNγ, IL-17) upregulated CD13 mRNA in FLS, which may contribute to the increased CD13 in RA synovium and synovial fluid. Inhibition of CD13 function by either inhibitors of enzymatic activity or anti-CD13 antibodies resulted in decreased growth and diminished migration of FLS. This suggests that CD13 may be involved in the pathogenic hyperplasia of RA FLS. This data expands potential roles for CD13 in the pathogenesis of RA

Metalloproteinases cleave CD13 from the surface of FLS.

<p>Five different protease inhibitors were added to FLS cultures covering all classes of proteases. <b>(A)</b> The only inhibitor to decrease shedding of CD13 into the supernatant of the cultures was GM6001. <b>(B)</b> No significant decreases were seen in cell lysate CD13 concentrations. Cultures were incubated with serum free media containing protease inhibitors for 48 hours: Pepstatin A (aspartic) 10μM, Aprotinin (serine) 100nM, Leupeptin (serine/cysteine) 10μM, GM6001 (metalloproteinase) 25μM, and E-64 (cysteine) 10μM. <b>(C)</b> TIMP-2 (0.6μg/ml) inhibited the secretion of CD13 from RA FLS while TIMP-1(0.6μg/ml) did not. We used FLS from 3 different RA patients. Secretion was measured by ELISA and optical density (OD) of the samples was measured. (n = 3) mean of % change ± SEM *p≤0.05 ***p≤0.0001</p

CD13 is found as a soluble protein and on extracellular vesicles.

<p><b>(A)</b> Supernatants from 3 flasks of RA fibroblasts were concentrated through a 30K centrifugal filter. RA synovial fluid was diluted with PBS (4:1), and 10mls of plasma was obtained from a healthy individual. Vesicle fractions were isolated from the samples by serial centrifugation. Supernatants from the final centrifugation were collected as the soluble protein fraction. The vesicle pellet was resuspended in 1ml PBS. CD13 was measured by ELISA and aminopeptidase activity was analyzed through cleavage of L-leu-AMC. Data is normalized to concentration in original fluid. mean±SEM n≥3 <b>(B)</b> Exosomes were lysed and purity was confirmed by western blot for flotillin-1 and CD9. Single bands appeared at expected sizes with a weak band for CD9 and a strong band for flotillin-1 confirming exosomes from FLS. <b>(C)</b> A discontinuous optiprep gradient was created in seven fractions from 1.268g/ml to 1.031g/ml. 500ul of the resuspended vesicles was layered onto the top of the gradient. The loaded gradients were centrifuged at 100kg for one hour. Fractions were collected in reverse. Fractions were washed in PBS at 110kg for 2hr and the pellets were resuspended in 500ul PBS. Soluble CD13 is present in the first supernatant separation, exosomes are in fractions 3–5, and other extracellular vesicles are shown from the other fractions. CD13 was observed in all three fractions. Data was converted to percentage of total fluid CD13. % total n≥3</p

CD13 is upregulated in FLS at the mRNA level but the effects on CD13 protein levels are varied.

<p>FLS were stimulated over a time course of 0–72 hours with IFNγ (1000U/ml), TNFα (10ng/ml), or IL-17 (10ng/ml). Cells were harvested and processed for either mRNA (A), surface expression (B), total cell lysate CD13 (C), or total CD13 in supernatant (D). mRNA was measured by qRT-PCR. CD13 was measured on the surface with anti-CD13 (1D7) and flow cytometry. Cell lysate and supernatant CD13 concentrations were measured by CD13 ELISA. Gating was done to isolate the major cell population and exclude debris and dead cells. Data is expressed as a ratio to unstimulated FLS at the same time point in either ΔΔCt normalized to GAPD (mRNA), mean fluorescent intensity corrected for background florescence with MsIg staiming (surface), or CD13 concentration in lysate or supernatant. A n = 3 B-D n = 1 *p≤0.05</p

MMP14 knockdown partially inhibits the shedding of CD13 from FLS.

<p><b>(A)</b> MMP14 mRNA was almost completely removed by transfection with MMP14 siRNA. ADAM10, ADAM15, and ADAM17 knockdown constructs each also almost completely removed their respective mRNAs with appropriate siRNA transfection. None of the siRNAs (MMP14, ADAM10, ADAM15, or ADAM17) had an off target effect on MMP1. n = 3 (<b>B)</b> Shedding of CD13 into FLS culture supernatant was inhibited by knockdown of MMP14. Further inhibition was seen with GM6001. CD13 in FLS lysate was not changed by knockdown of MMP14, and significantly increased in GM6001 treated FLS. FLS were grown to confluence then transfected with siRNA using an Amaxa nucleofector kit. Cells were grown to 75% confluence then switched to serum free growth media (Peprogrow) supplemented with 1ng/ml IL-1 and 10ng/ml TNFα for 48 hours with or without 25μM GM6001. <b>(C)</b> Cells were incubated for 1hour at room temperature with anti-CD13-FITC (1D7) 1μg/100μl and anti-MMP14-PE (128527) at 1.67μg/100μl. Figures shown were taken at 1000x. The four panels show: top left, MMP14 alone (red); top right, CD13 alone (green); bottom left, co-localization (co-localization in yellow); and bottom right, co-localization analysis by ImageJ (white = overlapping red and green pixels). All images were background corrected using DAPI alone and MsIg-FITC and MsIg-PE for threshold limits. Representative of n = 6 *p≤0.05 **p≤0.005 ***p≤0.0001 mean±SEM (n≥2)</p