| Abstract | Ozljede i oštećenja zglobne hrskavice vrlo su česte u ortopediji i sportskoj medicini,
a svako takvo oštećenje predstavlja rizik za rani razvoj osteoartritisa, uz značajno
smanjenje kvalitete života i povećanje troškova zdravstvene zaštite. Iako se trenutačno
korištenim metodama liječenja oštećenja hrskavice postižu vrlo dobri klinički rezlutati u
smislu smanjenja bolova i poboljšanja opsega pokreta, niti jedna metoda nije do sada
uspjela u potpunosti regenerirati normalnu hijalinu hrskavicu. Upravo zbog toga,
istraživanja novih mogućnosti liječenja oštećenja zglobne hrskavice predstavljaju izazov
podjednako i za kliničare i za one koji se bave bazičnom znanošću. Temeljna hipoteza
ovog istraživanja jest da je uporabom adenoviralnog vektora koji nosi gen za kodiranje
transformirajućeg faktor rasta-β1 (TGF-β1) moguće uspješno transducirati stanice
autologne koštane moždine, koje se potom u obliku ugruška implantiraju na mjesto
hrskavičnog oštećenja, a novostvorena hrksavica je kvalitativno bolja od one stvorene bez
uporabe genske terapije, odnosno od one stvorene samo umetanjem nepromijenjenog
uguška. Ciljevi ovog istraživanja bili su dokazati translacijsku vrijednost predložene
metode, analizirati učinke transdukcije autologne koštane moždine faktorom rasta TGF-β1
na obnovu hrskavičnog miljea u oštećenju, i to procjenom histoloških, biokemijskih i
biomehaničkih parametara. Pored toga cilj je bio utvrditi moguće „istjecanje“ virusnog
vektora u stanice sinovijalne ovojnice. Nakon pribavljenog odobrenja Etičkih
povjerenstava, u istraživanju su korištene ovce (n=28) u dobi od 1 do 3 godina. Na
medijalnom kondilu femura učinjeno je djelomično hrskavično oštećenje upotrebom
instrumenta za mozaikplastiku, u koje je potom transplantiran ugrušak autologne koštane
moždine. Ugrušak je u odgovarajućim skupinama prethodno genski promijenjen
upotrebom adenoviralnog vektora, odnosno transplantiran je nepromijenjen u kontrolnoj
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skupini. Metodom slučajnog odabira životinje su podijeljene u 4 skupine: CON (kontrolna
skupina, prazno oštećenje, n=6), BMC (pozitivna kontrola, transplantiran nepromijenjen
ugrušak, n=6), GFP (pozitivna kontrola, transplantiran ugrušak prethodno transduciran da
proizvodi GFP, n=6), te TGF (ispitivana skupina, transplatiran ugrušak prethodno
transduciran da proizvodi TGF-β1, n=10). Zdrava hrskavica iz kontralateralnog koljena
svake životinje korištena je kao kontrola ispitivanim skupinama. Životinje su žrtvovane
prema protokolu istraživanja nakon 6 mjeseci, a nakon žrtvovanja učinjena je histološka,
biokemijska i biomehanička analiza novostvorenog tkiva. Rezultati provedenog istraživanja
potvrdili su postavljenu hipotezu, te pokazali kako je primjenom predloženog ex vivo
protokola za upotrebu genski promijenjenog ugruška autologne koštane moždine moguće
dobiti hrskavični reparat zadovoljavajuće kvalitete, a uporaba faktora rasta TGF-β1
dodatno poboljšava kvalitetu novostvorenog tkiva. To je razvidno iz analize histoloških
preparata koji pokazuju da je u svim tretiranim skupinama došlo do stvaranja reparata,
osim u kontrolnoj skupini s praznim oštećenjem. Rezultati biokemijske analize pokazuju
optimalan udio glikozaminoglikana i kolagena tipa II jedino u skupini tretiranoj s TGF-β1.
Analiza biomehaničkih parametara IT AFM metodom otkriva kako je na mikrometarskom
nivou reparat hrskavice iz TGF skupine najsličniji nativnoj hrksavice, dok je na
nanometarskom nivou rezultat nešto lošiji obzirom na visoki ukupni udio kolagena i nizak
udio vode. PCR analiza potvrdila je odsutstvo virusa iz svih analiziranih uzoraka
sinovijalne ovojnice. Na osnovi rezultata provedenog istraživanja može se zaključiti kako je
primjenom ugruška autologne koštane moždine moguće dobiti zadovoljavajuć reparat
hrskavice u svim promatranim skupinama. Dodatna poboljšanja u kvaliteti reparata
moguće je ostvariti adenoviralnim prijenosom gena za kodiranje TGF-β1 koji se luči unutar
samog ugruška, te in situ usmjerava i regulira hondrogenezu. |
| Abstract (english) | Articular cartilage injuries are very common in orthopaedic surgery and sports
medicine, and often, with time they lead to premature osteoarthritis, and consequently a
decrease in quality of life and increase in costs of health care. Although mentioned
procedures produce good clinical result in terms of pain relief and improvement of joint
function, none of the currently used methods results in regeneration of healthy hyaline
cartilage. Due to that fact, numerous preclinical and clinical studies are trying to overcome
existing problems and suggest novel, improved methods to treat cartilage defects. Central
hypothesis of the proposed study suggest that is possible to use adenoviral vector carrying
gene for coding TGF-β1 to transduce autologous bone marrow clot which is subsequently
implanted into partial-thickness cartilage defects, and the newly formed tissue within the
defect is qualitatively superior to the one formed without gene therapy. The aims of the
proposed study are to: evaluate translational value of the proposed method, to analyze
effects of the transduction with TGF-β1 on restoration of cartilage by evaluation
histological, biochemical and biomechanical parameters. Additional aim was to determine
if there is a vector “leakage” from the clot into the surrounding synovial tissue. Upon
obtaining local Ethical Committee approval, 28 skeletally mature sheep (1 to 3 years old)
were used in this study. The sheep were randomly assigned to one of four groups. Partialthickness
chondral defect was made on the weight-bearing surface of the medial condyle
with standard mosaicplasty instrument, and corresponding bone marrow clots were
carefully inlaid into the defects without material fixation. In the bone marrow clot (BMC)
group (n=6), the sheep were implanted with untreated autologous bone marrow clot that
was aspirated from iliac crest of respected animal. In the bone marrow transduced with
Ad.GFP (GFP) group (n=6), the sheep were implanted with autologous bone marrow clots
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genetically modified to over express green fluorescent protein (GFP). In the bone marrow
transduced with Ad.TGF-β1 (TGF) group (n=10), the sheep were implanted with
autologous bone marrow clots genetically modified to over express transforming growth
factor-β1. Six untreated sheep served as a control (defect without implant). Undamaged
control articular cartilage also was taken from the medial condyle of the contralateral knee
joint. All sheep were euthanatized 6 months after surgery, and histological, biochemical
and biomechanical parameters of the newly formed tissue from the defect were analyzed.
The results confirmed the central hypothesis, as they showed that it is possible to use
abbreviated ex vivo protocol for genetical modification of autologous bone marrow clots to
produce repair tissue within the defect, and the use of TGF-β1 additionally improves the
quality of newly formed cartilage. Histological analysis showed that new tissue formed in
all the treatment groups except in the empty controls, and the biochemical analysis
showed optimal content of glycosaminoglycans and collagen type II only in TGF treated
group. IT AFM revealed that, on micrometer scale of tissue organization, newly formed
cartilage from TGF treated group has biomechanical properties that are the closest to the
native cartilage. On nanometer scale, the results are suboptimal due to high overall
collagen content and low water content. PCR analysis of the synovial lining tissue could
not detect residual presence of vector in none of the experimental groups. The results
suggest that proposed method is a single-step procedure that can be easily implemented
in standard clinical settings, and avoids the use of expensive in vitro production of
autologous, engineered tissues. It was possible to produce cartilage tissue of satisfactory
quality, and further improvements may be obtained with adenoviral transfer of TGF-β1 that
is secreted within the clot, and it stimulates and regulates the chondrogenesis process in
situ. |
