Mesenchymal stem cells and collagen patches for anterior cruciate ligament repair

Abstract

CG and NC are both cyto-compatible with primary MSCs and TCs; TCs seemed to perform better on these collagen patches than MSCs.

Biologic response of human anterior cruciate ligamentocytes on collagen‐patches to platelet‐rich plasma formulations with and without leucocytes

“…On the same day tissue was cut into ∼3 mm 3 pieces, washed with 1× phosphate buffered saline (PBS) and digested overnight in 230 U/mg collagenase 2 (Worthington, UK) in an incubated humidified environment (37°C, 5% CO 2 , 95% humidity), under constant shaking at 10 RPM. On the next day, the released cells were filtered with a 100 µm cell strainer (BD Falcon, Switzerland) and seeded at a density of 1,000 cells/cm 2 for monolayer expansion in high glucose Dulbecco's Modified Eagle's Medium (HG‐DMEM, Gibco, Switzerland) supplemented with 10% fetal calf serum (FCS, Sigma–Aldrich, Switzerland) and 1% penicillin/streptomycin (P/S, Sigma–Aldrich) as previously described …”

“…FDA‐approved collagen type I/III patches (ChondroGide™, Geistlich, Wohlhusen) were cut with a six mm biopsy punch (Kaimedical, Polymed Inc., Glattburg, Switzerland) and placed onto the agarose. 3 × 10 4 LCs ( N = 5 donors) at passage two were seeded on the rough side of collagen patches . All experimental groups were run in biological replicates n = 2 per donor.…”

“…Resazurin Sodium Salt (Sigma–Aldrich) was added to each well of the 12‐well plate (Falcon. BD, Allschwil, Switzerland) and wells were incubated at 37°C, 5% CO 2 for 3 h on a shaker at 10 RPM as previously described . When resazurin enters the cell it will be reduced to its red fluorescent form resorufin by mitochondrial enzymes.…”

“…1,9‐Dimethyl‐methylene‐blue chloride dye (DMMB, Sigma–Aldrich) binds to GAG and precipitates and consequently absorbance can be measured to ascertain the GAG content. Bovine chondroitin sulfate (Sigma–Aldrich) served as a standard . Acid hydrolysis and reaction with 4‐dimethylamino‐benzaldehyde (Sigma–Aldrich) and chloramine‐T (Sigma–Aldrich) was used to determine HYP content which was read at an absorbance at 560 nm.…”

“…Total RNA was quality‐checked for integrity and purity by Experion™ automated electrophoresis System (Bio‐Rad, Reinach, Switzerland). Total RNA (100–200 ng) was used for reverse‐transcription (RT) and subsequent PCR (real‐time‐qPCR) using gene‐specific primers . Oligonucleotide primers (all from Microsynth, Balgach, CH) were designed with Primer Beacon ® Designer™ Software (Premier Biosoft Inc., Palo Alto, CA) using nucleotide sequences taken from the GenBank database.…”