in companion animals , the healing of large skin defects resulted from traumatic accidents can be challenging to treat since they need a complex management consisting of accurate debridement of damaged and necrotic tissues , local and/or systemic infection control , protection of the underlying tissues and induction of cutaneous tissue regeneration 1,2 . to this aim , surgical tissue reconstruction ( i.e. , skin graft ) and medical treatments are often associated to reach complete anatomical and functional recovery of the damaged area , reducing debilitating side effects , and pain .
the present work describes a successful therapeutic approach based on the synergic application of platelet - rich plasma ( prp ) and autologous mesenchymal stem cells ( mscs ) in an adult labrador male dog .
the patient was involved in a traumatic accident that caused a very  large tissue defect ( approximately 30    35  cm , width    length ) of the superior area of the left thigh , that could not be resolved by surgery .
a 1-year -old labrador male dog , weight 33  kg , was referred about 812  h after being struck by a train .
the dog was conscious , well oriented in space and cooperating , but presented an extensive loss of skin tissues of the  superior area of the left thigh ( approximately 30    35  cm , width    length ) ( fig.1 ) .
the lesion was classified as a class 2 avulsion injury , 612  h old , with large loss of integumentary structures 1 .
furthermore , the dog presented fractures of the coccygeal vertebra with partial avulsion of the tail , bilateral lesions of the achilles tendons , a fracture of the left calcaneal bone , and several cutaneous wounds .
the animal did not show neurological deficits , but exhibited orthopedic deficits associated to the bilateral tendon injuries .
in particular , the first orthopedic evaluation showed the presence of lameness ( grade 3 ) of the hind limbs .
edema of both achilles tendons was present at the insertion to os calcaneus , where a 2  cm linear skin wound was present bilaterally .
a faster tissue growth was observed where wound margins strongly adhered to the underlying tissue . at day 90
the first day a lavage of the joints with physiological saline was performed , the left calcaneal bone fragment was removed , and the skin wounds were closed .
both lesions of the achilles tendons were 1  cm in length and 0.5  cm in depth , parallel to the tendon fibers .
we decided not to close the tendon lesions , to avoid fibrotic scar formation , but instead to proceed with a regenerative therapy ( see below ) .
no bandage , casting or external fixation was performed . with regard to the main lesion of the left hind limb ,
after the evaluation of the elasticity of the safe surrounding skin , we decided to perform a two - step surgery .
we first opted to close the wound with the surrounding skin , to reduce as much as possible the tissue exposition , thus creating a suitable environment for second intention healing of the residual exposed part of the wound .
a second surgery to perform a rotational flap or , alternatively , a skin graft to completely close the wound was scheduled 2030  days later 3,4 .
moreover , it was decided to proceed with the amputation of the tail . the fifth day after surgery ,
diffused necrosis of the edge of the skin , and the presence of purulent material were observed , thus we decided to remove the necrotic and contaminated tissue .
although the patient was a large size breed , he weighed only 33  kg and there was not enough skin available to perform a free skin graft .
in particular , we opted for a biologic and cellular therapy , to promote second intention healing with open wound management , based on the application of platelet - rich plasma ( prp ) and adipose tissue - derived mesenchymal stem cell ( mscs ) .
we reasoned that the association between mscs and prp could provide an appropriate set of biological mechanisms to speed up tissue growth , improve the quality of tissue regeneration , and control infection .
materials and methods used for the preparation of mscs and prp are described in appendix .
prp was applied either by dripping or spraying over the wound surface ( every 4872  h ) , as well as by injecting the platelet concentrate along the attached wound edge ( every 46  days ) .
mscs were applied by spraying the cells over the wound surface ( five applications at day 11 , 17 , 23 , 31 , 41 ) .
for each cell application , an aliquot of the cells was sprayed on a culture dish and growth in dmem to confirm their vitality ( fig.2 ) .
the lesions of the achilles tendons were treated by injecting under ultrasonographic guidance a single dose of 2    10 mscs resuspended in 1  ml of prp inside the lesions 5,6 . since the cutaneous defect was extremely extended , a wound managing strategy and planning was decided based on the  time system  7 .
the medication of the wound was performed daily , starting with the removal of old bandage , followed by the irrigation with lactated ringer s solution , debridement of the necrotic tissue , and application of prp and/or cell therapy .
the hydration of the wound was ensured by means of connettivina ( fidia farmaceutici , padova , italy ) or hypermix bandage ( ri.mos .
when prp or cells were not employed , hypermix oil and hypermix dressing were applied , to protect the tissue .
no antibiotic or disinfectant was applied locally . to prevent the movement of the bandage , a variation of  tie - over dressing / bandage technique  was applied 8 .
sprayed mscs culture . to assess mscs viability and replication after spraying , an aliquot of cells was cultured in dmem for 72  h. prp induced a partial gelation of the culture medium , therefore cells distributed and grew on different focal planes .
systemic antibiotic therapy was administered as follow : cefazolin ( cefazolina teva , milano , italy ) 30  mg / kg intravenous every 8  h from day 1 to 15 ; cefadroxil monohydrate ( cefa cure tabs , intervet italia , milano , italy ) p / o 20  mg / kg from day 16 to 30 ; marbofloxacin ( marbocyl vetoquinol , forl , italy ) 2  mg / kg from day 31 to 60 and amoxycillin and clavulanic acid ( synulox pfizer , roma , italy ) p / o from day 61 to 80 .
the huge area of the wound and rare episodes of fever in the first weeks suggested the use of the antibiotic therapy .
anyway , no signs of bacterial infection were observed after the onset of regenerative therapy .
analgesic therapy was administered with tramadol ( altadol formevet , milano , italy ) 2  mg / kg , every 8  h for the first 15  days .
anti - inflammatory therapy was given with carprofen ( rimadyl pfizer , roma , italy ) 2  mg / kg p / o , for 30  days , while gastric protection was provided with omeprazolum ( omeprazolo laboratori alter , milano , italy ) 10  mg p / o . before the daily care , for the first 20  days
, the animal was sedated with butorphanol ( butorphanol nargesic acme , reggio emila , italy ) 0.2  mg / kg and dexmedetomidine ( dexdomior orion pharma , milano , italy ) 5 microgr / kg ; afterwards , since the dog did not exhibit pain sensitivity , the sedation was suspended .
a granulation tissue was visible after 4  days following the first prp application . for a better perception of the speed of new tissue growth , as well as of the reduction in the exposed area
, we measured every day the amplitude of the wound , in the same position .
interestingly , the curve depicted in fig.3 fits a hyperbolic curve as described by cukjati et  al .
the ratio tissue growth / time reduced gradually during the period , with a fast growth during the first period ( about 2.5  cm / day in the first month ) and then a gradual fall during the remnant days .
a complete closure of the wound occurred 3  months after the start of the regenerative therapy ( fig.3 ) .
the wound never presented signs of infection or necrosis during and following the regenerative therapy .
the lameness of the hind limbs and the edema of the achilles s tendon disappeared 20  days after intra - lesional application of mscs and prp .
advancement of the wound margins , identified by the ls ( lesion size ) line , as a function of time .
the graph fits with a hyperbolic curve with a faster healing rate during the first weeks .
light blue rectangle indicates the duration of prp treatment ; red arrows indicate the msc treatments .
in recent years , mscs and platelet - derived products have gained a strong attention in veterinary medicine since they are supposed to enhance tissue regeneration and wound healing 10 .
although a definitive knowledge about the molecular and cellular mechanisms through which prp and mcs exert their effects is still lacking , several clinical and experimental reports confirm their efficacy for the treatments of patients both in veterinary and human medicine 1114 .
recently , a positive effect of the application of mscs in healing of experimentally induced skin wounds has been reported in the dog 15 .
mscs respond to wound healing cytokines and migrate to region of inflammation creating a favorable immune environment 16 .
mscs secrete a plethora of effector molecules and produce microvesicles containing signaling proteins , mrnas , mirnas 17,18 . by means of these mechanisms ,
they reprogram resident immune cells , control inflammation , activate tissue progenitors cells , promote angiogenesis .
mscs also regulate leukocytes ( t and b cells , natural killer cells ) function and activate resident macrophages to produce anti - inflammatory cytokines .
. in particular , cox2 and pge2 upregulation are probably key elements of immunomodulatory mechanisms of the mscs as well as of their antiscarring effects 16 .
large cutaneous lesions , where poor blood supply , tissue necrosis , excessive scarring , inflammation , and bacterial contamination are possible complications , would take significant advantages from the application of regenerative therapies based on the synergic action of prp and mcs 16,1922 . here , we report the case of a particularly large cutaneous lesion in a dog hit by a train . the area involved in the lesion
after the failure of the first surgery aimed to close the wound with the surrounding skin , the physical conditions of the patient were serious , although not critical .
furthermore , although the patient was a labrador , he weighed only 33  kg and there was not enough skin to perform a free skin graft .
since the main lesion was particularly extended to reach a homogeneous distribution of the therapeutics , we sprayed the whole injured area with both prp and mcs .
parallel in  vitro cell cultures showed that sprayed mscs could survive and growth inside a prp - derived gel ( fig.2 ) , thus supporting this route of therapy administration .
connettivina and hypermix dressing were used to improve tissue hydration when prp and mscs were not applied .
the application of prp and autologous adipose tissue - derived mscs probably contributed to the positive outcome , inducing a prompt formation of granulation tissue , a strong adhesion of the wound edges to the underlying tissue and a rapid tissue growth , starting from wound margin .
furthermore , no sign of excessive scarring , tissue contraction , and fragile re - epithelialization , sometimes associated to second intention healing , was observed .
in addition to the amelioration of the tissue growth , prp and mscs probably contributed to a faster wound healing providing an antibacterial and anti - inflammatory environment 23,24 .
antimicrobial properties are probably do to their capacity to reduce inflammatory response and to increase macrophages and monocytes phagocytosis 27,28 .
furthermore , mscs secrete gm - csf , a cytokine that exert significant antimicrobial activity 27 . as a matter of fact , following the regenerative therapy , no sign of bacterial contamination was evidenced , even though the exposed area was vast and extended to the perianal region ( fig.1 ) .
these results suggest that mscs and prp are indeed an effective therapeutic option to manage soft tissue wounds where a large amount of tissue is destroyed , especially when surgery alone can not guarantee satisfactory results .
regenerative therapy can be considered by surgeon both to improve the quality of tissue regeneration and to speed up the wound healing process .
finally , this report outlines that in clinical veterinary medicine , effective collaboration of the owner , and a highly collaborative approach between clinical and biomedical staffs , are essential to lead to a successful healing of complicated wounds and ensure  the return of the patients to an excellent quality of life .
mesenchymal stem cells preparation : for the preparation of mscs , three grams of healthy subcutaneous adipose tissue were collected along the edge of the wound , minced into small pieces under sterile conditions , and washed several times with phosphate buffer saline solution ( pbs ) containing penicillin ( 1000  iu / ml ) and streptomycin ( 100  mg / ml ) ( sigma - aldrich corp . , st .
the sample was then digested in 15  ml of a 0.75  mg / ml solution of type i collagenase ( sigma - aldrich corp . ) dissolved in dulbecco s modified eagle s low glucose medium ( dmem ) ( lonza , inc .
after digestion , the remaining tissue fragments were removed , while the cell suspension was filtered through a 50  m nylon mesh and then centrifuged at 250  g for 10  min . the supernatant was removed and the cell pellet resuspended in dmem containing 10% v / v fetal bovine serum ( fbs ) ( lonza , inc . ) , 1000  iu / ml penicillin and 100  mg / ml streptomycin .
finally , the cells were seeded in 25  cm tissue culture flasks at the concentration of 5.0    10  cells / cm , and incubated at 37c in 5% co2 .
cell viability was always > 90% . to assess mscs viability and replication after spraying ,
an aliquot of the cells used for the therapy was sprayed on a cell culture dish and was cultured in dmem containing 10% v / v fetal bovine serum ( fbs ) and antibiotics for 72  h. the expression of cell surface markers cd13 , cd34 , cd44 , cd45 , cd90 , cd105 has been assessed by rt - pcr ( data not shown ) .
cell cultures were positive to cd44 , cd90 , and cd105 , and negative to cd45 and cd13 expression .
cells were also positive to cd34 expression , as often reported for adipose tissue - derived mscs 29 .
furthermore , cells were positive to adipogenic , osteogenic , and chondrogenic differentiation ( appendix fig.1 ) .
platelet - rich plasma preparation : platelet - rich plasma ( prp ) was obtained from autologous venous blood collected with citrate - phosphate - dextrose . fresh blood samples ( about 2530  ml )
were centrifuged at 150  g for 30  min at 4c in a 50  ml tube .
the erythrocyte fraction was discarded , while the plasma , enriched with platelets , was further centrifuged at 800  g for 15  min .
the platelet pellet was collected and the platelets were counted ( cell - dyn 3500r haematology analyser , abbott , roma , italy ) and diluted at a final concentration of 1.0    10  cell / ml .
platelet - rich plasma was applied soon after preparation ( prp ) , or as platelet - rich plasma lysate ( prplys ) . in this case
 key clinical messageextensive full - thickness skin wounds are quite common in domestic animals . in these report , 
 following the failure of reconstructive surgery , adipose tissue - derived mesenchymal stem cells , and platelet - rich plasma were successfully used in a dog to improve speed and quality of skin tissue healing , avoiding suffering , and debilitating effects . 
Introduction
Case History
Assessment of Therapeutic Options and Treatment
Outcome and Follow-up
Discussion
Conflict of Interest
None