Page 3 - Gastric pentadecapeptide BPC 157

918 M. Siuresinic el u(. / J/ournul of Or[hopudic Riwurch 21 (2003) 976-983

a pool of tissue by consecutive enzymatic digestion of matrix (first definitive indicator of advanced recovery of the Achilles
hyaluronidase, second trypsin and third collagenase). The cells were tendon function going on after transection [2]. Sup-
seeded in plastic tissue culture flask (Greiner, Germany) and incubated portingly, AFI-values are in generally different (different
in RPMl 1640 medium supplemented with 10'%) fetal calf serum (FCS,
Sigma, USA) at 37 "C in humidified air atmosphere with 5% COz for AUC (cm') (median [minimum to maximum]) (i.e.,
seven days before being used for the experiment. Thus obtained semi- healthy -127 [-170.2 to -8.71, controls with transected
confluent culture was harvested by trypsin, viability of cells was vali- tendon -906 [- 1030 to -782.5]*, gastric pentadecapep-
dated by trypan blue exclusion and they were seeded in 96-well
microcyto plates (Greiner, Germany) at 1 O4 cells/well seeding density. tide BPC 157 pg -597.5 [-645.5 to -580.5]*', ng -696.5
1-743 to -676]", pg -897.5 1-1026 to -780.51'; *VS.
Cells treuted wirh BPC 157 or TGF-/I1 uncilor HNE in ilir prescvicr o/ healthy p < 0.01, 'vs. control with transected tendon
.seruni p < O.Ol), and particularly, since day 3, AFI-values are
Two hours the cells were seeded in microwell plates, and then to
discriminate between pre-treatment (i) and post-treatment (ii)-condi- higher in pentadecapeptide BPC 157 rats. Finally, these
tions, (i) BPC 157 (as 2 pglml final concentration) or TGF-PI (at 10 ng/ values reached those otherwise present in healthy rats.
ml final concentration) or (ii) HNE (at 1.56 mg/ml final concentration) Accordingly, an early effect is identified, at a critical one
was added. Subsequently, the cells were cultured for next 2 h when (i)
HNE (at 1.56 mg/ml final concentration) or (ii) BPC 157 (as 2 pglml day period.
final concentration) was added. Control cell cultures were incubated Pentadecapeptide BPC 157-treated rats have more
either in medium without HNE or without peptide, or in plain medium mononuclears (pg group 17 [10-36], p < 0.01, and ng
only. HNE was prepared from HNE-DEA. Each treatment was car-
ried for quadruplicates of cell cultures. group 17 [lo-331, p < 0.01, but not pg group 9 [1-17],
p > 0.05) than controls (9 [O-IS]), and less granulocytes
Scrurn tl~~prioc~cl conditions (pg group 12 [7-201, p < 0.01, and ng group 12 [6-191,
To increase sensitivity to the growth factors [4], the cells were kept p < 0.01, but not pg group 25 [7-901, p > 0.05) than
in the presence of only 0.1'%1 FCS instead of 10% FCS for 24 h before
and during the treatment with BPC 157 (2 pglml final concentration), controls (34 [ 13-99]), subsequently presenting with ret-
TGF-PI (10 ng/ml final concentration) and/or HNE (1.56 mg/ml final iculin fibers denser and more regular distribution, higher
concentration). In combined studies, the aldehyde was applied only fibroblasts number, denser and a more regular distri-
after the peptide.
For both treatment protocols incorporation of methyl-2H-thymi- bution of collagen fibers following the distribution
dine (Amersham, UK) was analyzed for the last 24 h incubation when of reticulin fibers.
the cells were harvested over glass filter using a cell-harvester system Along with positively influenced formation and dif-
(Skatron. Norway) and the intensities of radioactivity of incorporated
'H-thymidine was determined in a j3-counter (LS3800, Beckman. ferentiation (maturation), and advancing tendon heal-
USA). ing, we note in pentadecapeptide BPC 157-treated rats
at very early intervals, on day 1, smaller size and depth
Siciti~iicuI unu1y.si.s of tendon defect compared to controls (pg group 2.7 mm
For in vivo studies Kruskall-Wallis and Mann-Whithney tests
were used for analysis, and data presented as median, minimum and [2.1-3.61, p < 0.01, ng group 2.7 mm [2.1-3.91, p < 0.01,
maximum (MED/MIN/MAX respectively). Changes in AFI were ex- pg group 6.9 mm [5.5-7.51, p > 0.05) and subsequently
pressed using AUC method (area under curve, cm', 1 cm y-axis rep- the reestablishment of full tendon integrity. Besides the
resents 10 AFJ units, I cm x-axis represents 1 day). Due to Bonferroni defect disappears in treated rats three days earlier than
correction. the significance of p values was interpreted according to the
number of compared groups (significant were values of p < 0.012 or in controls, at the end (14 days post-injury), formed
less for five groups comparisons, p < 0.016 or less for four groups white tendon tissue with a non-disrupted continuity
comparisons and p < 0.05 for two groups comparisons. Likewise, in consistently in treated rats contrasts with transparent
vitro results were analyzed using Mann Whitney test and the values
of p were regarded statistically relevant as mentioned above. tissue and still obvious distal end present in controls.
Failure load, failure load per area, Young's modulus of
elasticity, all markedly increased, signify in pentadeca-
Results peptide BPC 157-treated rats a steady healing im-
provement and correspondingly increased strength of
Evaluation oj deficit und recovery of' Achilles tendon tissues during Achilles tendon healing. Finally, the
fo Ilo 1 ring t ransect ion values otherwise present in healthy rats were reached.

Impaired healing of transected right Achilles tendon Cell cultures
in controls follows the course and the figures commonly
reported by others (i.e., [2]). Large tendon defect ap- Ce11.r treated with BPC 157 or TGF-PI andlor HNE in the
peared between the cut ends of Achilles tendon, mea- presence of serum
suring (median [minimum-maximum]) 7.6 mm [6.2-8.31 BPC 157 itself did not effect the growth of cul-
immediately after transection and 7.4 mm [6.2-7.91 on tured tendocytes. TGF-PI caused no effect on tendocyte
day I. growth (Fig. 4). However, BPC157 interfered with the
Noteworthy, positive outcome in pentadecapeptide effects of HNE in vitro on the growth of tendocytes cells
BPC 157 rats (pg- or ng-regimen) is supported by func- (Fig. 5). HNE reduced 3H-thymidine incorporation into
tional, biomechanical, microscopical and macroscopical cultured tendocytes cells. BPC 157 present before HNE
findings (Table 1, Figs. 1-3). AFI-values are the most reverses HNE-growth inhibition is reversed into a