Sr Rathbone

Biocompatibility of polyhydroxyalkanoate as a potential material for ligament and tendon scaffold material

There is a strong need for new biodegradable materials that are suitable for scaffolds in tissue engineering of tendons and ligaments. In many cases, quick degradation rates are favorable, however, with respect to ligament and tendon replacement, slowly degrading polymers are clearly favored. Prime candidates are members of the large class of polyhydroxyalkanoates (PHAs), which are thermoplastic/elastomeric biopolyesters that are slowly degraded by surface erosion. Moreover, their physico-mechanical properties can be tailored during biosynthesis in bacteria or by chemical modifications. They may be spun into fibers, coated on surfaces or be part of composites. This study has investigated the biocompatability of seven different thermoplastic or elastomeric PHAs using L929 murine fibroblast cells. Cell viability and proliferation over 7 days was analyzed with live/dead staining and a picogreen assay. In addition, extracellular matrix production was measured with the hydroxyproline assay after 14 days. It was found that cell attachment to the PHA film ranged from 85-99% after 7 days. Three PHA films (PHBV (92/8), PHOUE-POSS and PHUE-O3) supported similar cell viability in comparison to the controls performed on tissue culture plastic (polystyrene), whereas the biomaterials (PHUA, PHUE, PHB and PHOUE) showed fewer viable cells than in controls. PHB, PHUE-O3, and PHBV with a water contact angle below 85 degrees supported a similar amount of collagen production in comparison to the tissue culture plastic controls. PHUA, PHUE, PHOUE, and PHOUE-POSS showed a decrease in collagen production in comparison to the controls after 14 days. Overall, PHB, PHBV, and PHUE-O3 demonstrated good performance with regards to potential use as a tissue-engineering scaffold.

Dynamic loading of electrospun yarns guides mesenchymal stem cells towards a tendon lineage

Alternative strategies are required when autograft tissue is not sufficient or available to reconstruct damaged tendons. Electrospun fibre yarns could provide such an alternative. This study investigates the seeding of human mesenchymal stem cells (hMSC) on electrospun yarns and their response when subjected to dynamic tensile loading. Cell seeded yarns sustained 3600 cycles per day for 21 days. Loaded yarns demonstrated a thickened cell layer around the scaffold׳s exterior compared to statically cultured yarns, which would suggest an increased rate of cell proliferation and/or matrix deposition, whilst maintaining a predominant uniaxial cell orientation. Tensile properties of cell-seeded yarns increased with time compared to acellular yarns. Loaded scaffolds demonstrated an up-regulation in several key tendon genes, including collagen Type I. This study demonstrates the support of hMSCs on electrospun yarns and their differentiation towards a tendon lineage when mechanically stimulated.

Most British Surgeons Would Consider Using a Tissue-Engineered Anterior Cruciate Ligament: A Questionnaire Study

Donor site morbidity, poor graft site integration, and incorrect mechanical performance are all common problems associated with autografts for anterior cruciate ligament (ACL) reconstructions. A tissue-engineered (TE) ligament has the potential to overcome these problems. We produced an online questionnaire relating to tissue engineering of the ACL to obtain input from practising clinicians who currently manage these injuries. 300 British orthopaedic surgeons specialising in knee surgery and soft tissue injury were invited to participate. 86% of surgeons would consider using a TE ACL if it were an option, provided that it showed biological and mechanical success, if it significantly improved the patient satisfaction (63%) or shortened surgical time (62%). 76% felt that using a TE ACL would be more appropriate than a patellar tendon, hamstring, or quadriceps autograft. Overall, most surgeons would be prepared to use a TE ACL if it were an improvement over the current techniques.

Cyclic tensile strain upon human mesenchymal stem cells in 2D and 3D culture differentially influences CCNL2, WDR61 and BAHCC1 gene expression levels.

It has been shown that tensile strain can alter cell behaviour. Evidence exists to confirm that human mesenchymal stem cells can be encouraged to differentiate in response to tensile loading forces. We have investigated the short-term effects of cyclic tensile strain (3%, 1 Hz) on gene expression in primary human mesenchymal stem cells in monolayer and whilst encapsulated in a self-assembled peptide hydrogel. The main aims of the project were to gain the following novel information: (1) to determine if the genes CCNL2, WDR61 and BAHCC1 are potentially important mechanosensitive genes in monolayer, (2) to determine if these genes showed the same differential expression in a 3D environment (either tethered to RGD or simply encapsulated within a hydrogel (with RGE motif)) and (3) to determine whether the mesenchymal stem cells would survive within the hydrogels over several days whilst enduring dynamic culture. In the monolayer system, real-time PCR confirmed CCNL2 was significantly downregulated after 1 h strain and 2 h latency (post strain). BAHCC1 was significantly downregulated after 1 h strain (both 2 h and 24 h latency). WDR61 followed the same trend in 2D culture. After 24 h strain and 2 h latency, BAHCC1 was significantly upregulated. We found that both types of peptide hydrogel supported viable mesenchymal stem cells over 48 h. Results of the 3D dynamic culture did not correspond with those of the 2D dynamic culture, where the BAHCC1 gene was not expressed in the 3D experiments. The disparity in the differential gene expression observed between the 2D and 3D culture systems may partly be a result of the different cellular environments in each. It is likely that cells cultured within an intricate 3D architecture respond to mechanical cues in a different and more complex manner than do cells in 2D monolayer, as is illustrated by our gene expression data.

3D sample preparation for orthopaedic tissue engineering bioreactors.

There are several types of bioreactors currently available for the culture of orthopaedic tissue engineered constructs. These vary from the simple to the complex in design and culture. Preparation of samples for bioreactors varies depending on the system being used. This chapter presents data and describes tried and tested methodologies for the preparation of 3D samples for a Rotatory Synthecon Bioreactor (Cellon), a plate shaker, a perfusion system, and a Bose Electroforce Systems Biodynamic Instrument for the in vitro culture of bone and ligament tissue.

Tissue Engineering of Ligaments

The main function of a ligament is to connect one bone to another bone across a joint, keeping them aligned to prevent abnormal motions and dislocations. The typical magnitude of force a ligament may experience during day-to-day activities varies. For example the anterior cruciate ligament of the knee can be exposed to daily tensile forces ranging between 67N for ascending stairs to 630N for jogging (Vunjak-Novakovic, Altman et al. 2004), whereas large loads, exceeding 1800N, can cause rupturing. Depending upon anatomical location and the extent of vascularisation, the ligament may or may not be capable of selfhealing after a rupture. Some of the most frequently ruptured ligaments occur in the knee joint, often through sporting activities such as skiing, football and basketball and the number of injuries are increasing each year (Cooper, Lu et al. 2005). Ninety percent of knee ligament injuries involve the anterior cruciate ligament (ACL) and medial collateral ligament (MCL) (Woo, Abramowitch et al. 2006). The MCL can self-heal, but the ACL cannot due to poor vascularisation. Because of this, alternative methods such as regenerative medicine have focused heavily upon the ACL with the aim of producing a fully functional tissue in vitro. Figures indicate that approximately 250,000 people are diagnosed with ACL injuries each year in the USA (Doroski and Brink 2007), and approximately 150,000 need to undergo surgical treatment, known as an ACL reconstruction (Cooper, Lu et al. 2005). If the rupture is not treated it can cause loss of function of the associated joint which can then lead to early development of osteoarthritis (Cooper and Bailey 2006; Gentleman, Livesay et al. 2006). The current gold standard procedure for an ACL reconstruction is surgical autografting. This involves using part of the patients own patellar tendon, hamstring or quadriceps to replace the ruptured ACL (Beasley, Weiland et al. 2005). However, these techniques cause donor site morbidity (Goulet and Germain 1997; Van Eijk, Saris et al. 2004; Cooper and Bailey 2006; Hairfield-Stein, England et al. 2007) which is associated with pain and a recovery period for the donor tissue site (Cooper, Lu et al. 2005; Hairfield-Stein, England et al. 2007). Generally 75-90% of patients have good or excellent long term success rates from these current grafting techniques (regarding functional stability and symptomatic relief upon return to normal activities) but unfortunately a substantial number of patients exist who have unsatisfactory results which could be attributed to graft failure (Vergis and Gillquist 1995). Some of these patients continue to endure pain, suffer from loss of motion secondary to the operative procedure and continue with recurrent instability (Vergis and Gillquist 1995), while others suffer from degenerative joint disease such as arthritis or

Optimizing Attachment of Human Mesenchymal Stem Cells on Poly(ε- caprolactone) Electrospun Yarns

Research into biomaterials and tissue engineering often includes cell-based in vitro investigations, which require initial knowledge of the starting cell number. While researchers commonly reference their seeding density this does not necessarily indicate the actual number of cells that have adhered to the material in question. This is particularly the case for materials, or scaffolds, that do not cover the base of standard cell culture well plates. This study investigates the initial attachment of human mesenchymal stem cells seeded at a known number onto electrospun poly(ε-caprolactone) yarn after 4 hr in culture. Electrospun yarns were held within several different set-ups, including bioreactor vessels rotating at 9 rpm, cell culture inserts positioned in low binding well plates and polytetrafluoroethylene (PTFE) troughs placed within petri dishes. The latter two were subjected to either static conditions or positioned on a shaker plate (30 rpm). After 4 hr incubation at 37 oC, 5% CO2, the location of seeded cells was determined by cell DNA assay. Scaffolds were removed from their containers and placed in lysis buffer. The media fraction was similarly removed and centrifuged – the supernatant discarded and pellet broken up with lysis buffer. Lysis buffer was added to each receptacle, or well, and scraped to free any cells that may be present. The cell DNA assay determined the percentage of cells present within the scaffold, media and well fractions. Cell attachment was low for all experimental set-ups, with greatest attachment (30%) for yarns held within cell culture inserts and subjected to shaking motion. This study raises awareness to the actual number of cells attaching to scaffolds irrespective of the stated cell seeding density.