Extraction and biomedical application of peripheral blood stem cells in sheep and horses

[摘要] SUPERFICIAL digital flexor tendon injury has a serious negative impact on thecompetitive horse industry. Injured horses require up to a year of rest for recovery andlikelihood of re-injury upon return to normal activity is as high as 80 %. Tendon healingrequires (a) production of collagen by fibroblasts, to provide tensile strength and elasticity tothe tendon, (b) minimisation of restrictive fibrosis, which compromises tendon glidingfunction and (c) minimisation of peritendinous adhesions. We review conventionaltreatments for tendon healing before exploring stem cell application as a therapeuticalternative. We promote the use of hematopoietic and mesenchymal stem cells derived fromadult peripheral blood - as opposed to bone marrow-derived stem cells or embryonic stem cellsources - and review published research output in this regard. In conclusion, we outline ourresearch objectives and present and discuss our results in the chapters that follow.Mononuclear cells - consisting of hematopoietic stem cells, mesenchymal stem cellsand leucocytes – were isolated from the peripheral blood of sheep and horses through redblood cell lysis and blood plasma extraction. Cell counts and propidium iodide dye exclusionviability tests were conducted on the cell pellets. Sheep sub samples were tested for CD45expression and horse sub samples for CD4 and CD11a/18 cell surface markers by flowcytometry for characterisation purposes. In both cases, separate sub samples were incubatedwith matched immunoglobulin (IgG) isotypes, conjugated to fluorescein isothiocyanate(FITC), to serve as controls. For the culture of mononuclear cells, 4.5 x 106 cells wereselected for autologous sheep injections, 3 x 106 CD45- cells for allogeneic sheep injections(the latter excluding leucocytes that may induce an immune response) and 72 x 106 cells forhorse injections. These cells were incubated with bromo-deoxyuridine (BrdU), cultured andsubsets were extracted for a second round of cell counts and viability tests before beingresuspended in blood plasma. For the horse samples an additional 1 x 106 mononuclear cellswere incubated until reaching 60 % confluence and tested for myogenic differentiation. Lowcell mortality and lack of fluorescence from IgG-FITC controls reflected effective protocolsand a lack of false positive results. The fact that the equine cell population differentiated intomyotubes verified the presence of mesenchymal stem cells in injections.We tested whether surgical incisions or collagenase injections best mimicked naturallyoccurring tendon injuries and compiled macroscopic and microscopic descriptions of tendoninjury sites at seven weeks post-injury. The superficial digital flexor tendons of 27 sheepreceived an incision, a collagenase injection or a saline control injection. After one week a number of sheep were sacrificed while the remainder received further saline treatment andwere sacrificed after another seven weeks. Tendons were examined through clinicalobservations, image analysis of maximum tendon diameter, mechanical testing andhistological sectioning of affected tissues. Collagenase-induced injury resembled tendonitismore closely than surgically-induced injury. Collagenase-injured tendons (a) inducedlengthier lameness in affected limbs, (b) were more swollen and difficult to palpate, (c)assumed the bow appearance characteristic of natural injury, (d) experienced extensivehaemorrhage due to collagen lysis, (e) had decreased elasticity and capacity to carry loads andstress, (f) displayed decreased stiffness due to collagen fibre disruption and (g) developedsevere inflammation. After seven weeks injured tendons displayed increased vascularisationin the areas of haemorrhage and in the adjacent collagen matrix. High inflammation rates andlow collagen levels however still persisted.Collagenase injections were used to induce tendonitis in the superficial digital flexortendons of 27 sheep. After one week these tendons received treatment with a control salinesolution, autologous peripheral blood mononuclear cells (MNCs) or allogeneic peripheralblood CD45- MNCs. Healing rates were compared after a further seven week period byconducting ultrasonographic evaluations, clinical observations, image analyses of maximumtendon diameter, mechanical tests and histological investigations. Tendons treated withMNCs displayed an improvement in echogenicity and fibre linearity, higher and moreorganised collagen levels, stronger mechanical properties and less swelling. Although theseimprovements were not always significant, they provided strong evidence to suggest markedhealing benefits over a longer time period.Collagenase injections were used to induce tendonitis in the superficial digital flexortendons of four horses. After one week these tendons received treatment with either a controlsaline solution or autologous peripheral blood mononuclear cells (MNCs). Healing rates werecompared after a further seven week period by conducting ultrasonographic evaluations,clinical observations, image analysis of maximum tendon diameter and histologicalinvestigations. Tendons treated with MNCs displayed significant improvements in fibrelinearity in the direct vicinity of the lesion, as well as recovery rate thereof, and experiencedless swelling when compared with their untreated counterparts. Healing trends suggestedthat, given a longer period of observation post-injury, more significant improvements maybecome apparent.Human adipose tissue is known be an easily accessible and high yielding source ofmultipotent mesenchymal stem cells. These stem cells could potentially be used for therapeutic advancement of tendon regeneration. Our first goal was to examine the in vitromyogenic differentiation potential of adipose-derived, adherent mononuclear cells (MNCs)from six adult sheep. The second goal was to characterise the population of cells isolatedthrough various available ovine specific, non-mesenchymal stem cell surface markers,namely, CD1, CD31, CD34 and CD45. After incubation, only four of the six MNC culturesstarted to proliferate. These four cultures all exhibited high myogenic differentiation ability.The isolated cell populations did not express any of the non-mesenchymal stem cell specificcell surface markers.In conclusion, our data suggests that peripheral blood stem cells and adipose-derivedstem cells are important candidate cell types for therapeutic application to improve tendonrepair in horses and sheep. Sufficient time must be allowed following injury and prior to stemcell treatment (at least one month) and a controlled exercise program should be followed posttreatment.A larger sample size is required and at least six months of recovery beforemacroscopic and histological repair can be analysed more accurately and conclusively.Ultrasonography should be carried out on a continuous basis, as it is a non-invasive method ofmonitoring change over time.

[发布机构] Stellenbosch University