Stem cell therapy applied for digestive anastomosis: Current state and future perspectives

Table 1 Kind of stem cells employed in published studies classified by their origin and type of transplant

Kind of stem cells employed
Myoblasts	Bone marrow MSCs	ASCs	Autologous/syngeneic	Allogeneic	Xenogeneic
ALLOG: 1	AUT: 1; ALLO: 2	AUT: 7; ALLOG: 4; XENOG: 2	8	7	2

Numbers indicate the number of published studies. MSCs: Mesenchymal stem cells; ASCs: Adipose-derived stem cells; AUT: Autologous; ALLOG: Allogeneic; XENOG: Xenogeneic.

Table 2 Systems utilized to apply stem cells

SCs delivery system
Biosutures	Local injection	Systemic injection	Local + systemic injection	Topical	Cell sheets	Gelatin sponge	Stent + mesh
2	71,2,3	1	1	24	4	1	14

¹One study associated thrombin and fibrin.

²One study compared local injection with and without fibrin glue.

³One study applied stem cells (SCs) in fibrin glue into a stablished fistula.

⁴One study compared topical versus stent and mesh with SCs.

SCs: Stem cells. Numbers indicate the number of published studies.

Table 3 Animal species employed in published preclinical studies

Type of animals
Rats	Pigs	Rabbits
12 (9 colorectal, 3 gastric perforation)	4 (small bowel and biliary anastomoses)	1 (esophageal fistula)

Numbers indicate the number of published studies.

Table 4 Overview and concise review of different published studies related to digestive anastomosis/perforations and stem cell therapy in animal models

Ref.	Ani-mal	N	Rando-mized	Anast/perf model and repair	SC doses and type	SC treatment	Compared to	Effect measure	Follow up	Principal results	Security concerns
Colon and colorectal anastomoses
Pascual et al[32]	Rats (BDIX)	40	No	Right colon section. Interrupted end-to-end	1.5 × 106 SYNG ASCs	20 biosutures	Conventional suture	Surgical evaluation (dehiscence, dilatation, obstruction, adherences). Bursting pressure; Histology	4, 7, 14, 21 d	Lower adhesion index at 4 d (P = 0.025) and 7 d (P = 0.006). No differences in the other outcome measures	No
Pascual et al[36]	Rats (BDIX + SD)	18	No	Identical to high risk: icodextrin. Identical	SYNG ASCs	6 biosutures + icodextrin	Conventional suture +/- icodextrin	Surgical evaluation (dehiscence, adhesion). Bursting pressure	4 d	No differences in dehiscence. Conventional sutures: icodextrin ↓ adhesion and MBP. Icodextrin: Biosuture ↑ MBP with equal adherences	No
Adas et al[37]	Rats (WI)	40	No	Ischemic: Left colon section + 4 cm vessel ligation. Interrupted end-to-end.	5 × 105 ALLOG BM-MSCs	20 local injection	Saline solution	Surgical evaluation. Bursting pressure. Hydroxyproline. Histology. SC tracing	4 and 7 d	No leakages, peritonitis, mortality. SCs ↑ MBP (2×) and hydroxyproline. Histology favourable for healing at both timelines. SC survive and proliferate	No
Yoo et al[38]	Rats (SD)	60	No	Ischemic: Left colon section + vessel ligation until > 50% flow reduction. End-to-end PLP.	1 × 106 ALLO ASCs	30 local injection + fibrinogen & thrombin	Ischemic anastomoses	Clinical follow-up: Surgical evaluationABP. Histology	7 d	ASCs: ↓ weight loss and earlier weight recovery; ↓ ileus, ulcers and strictures. ↑ MBP. Histology: SCs ↓ inflammation and ↑ collagen and microvascular density.	No
Adas et al[39]	Rats (WI)	40	No	Ischemic: Left colon section + 4 cm vessel ligation. End-to-end interrupted	1 × 106 ALLOG BM-MSCs	20 systemic injection	Saline solution	Surgical evaluation. Bursting pressure. Hydroxyproline. Histology. SC tracing	4 and 7 d	No leakages, peritonitis, mortality. SCs ↑ MBP (43%) at 4th but not 7th day. SCs ↑ SS hydroxyproline. Histology SS favourable for healing (4, 7d). SC Survive and proliferate	No
Sukho et al[40]	Rats (WI)	60	Yes	Partial right colectomy. Insufficient end-to-end (5 stitches).	XENOG human ASCs	30 sheets wrapping anastomosis	Insufficient anastomosis	Follow-up: Macroscopic evaluation. ABP. Histology	3 and 7 d	ASCs ↓ dehiscence (14% vs 71%) at 3 d, abscesses at 7 d and abdominal adhesions at 3 d. ABP ↑ 3 to 7 d, but NSS differences between groups. Labelled cells detected at both periods. Histol: SCs ↑ CD3+ and maintain CD163+ cells at 7 d.	No
Van de Putte et al[43]	Rats (SD)	24	No	IrradiatedColon section. Interrupted end-to-end	5 × 106 IV and 2.5 × 106 local. ALLOG ASCs	10 local injection + IV -7, 10, 20 d	Conventional anastomosis. Irradiation + anast + PBS	PET. Colonoscopy. Histology	4 wk	PET: preop IV ASCs ↓ activity to non-irradiated level. No differences at 4 wk. Colonoscopy: ASCs ↓ necrotic tissue and fibrin and bleeding (??P). Histology: SS ASCs ↓ ulcerated area and ↑ number vessels. ↑ M2 macrophages (??P).	0/3/3 deaths. No ASCs related
Alvarenga et al[44]	Rats (WI)	61	Yes	TNBS colitis. Left colon section. Ent-to-end interrupted	2 × 106 ALLOG ASCs	15 instillation over anastomosis	G1, TNBS colitis. G2, Laparotomy. G3, colitis + anast. G5, colitis + anast + CS	Follow-up: Macroscopic. Histology, IHQ, RNA	7 d	ASCs ↓ mortality to 0% compared to G3/G5 and local complications to 0%. ASCs: ↓ inflammation, tissue damage, myeloperoxidase activity, CD4+ and ED1+ macrophages, apoptosis; and ↑ epithelization (vs G5). ASCs: ↓ IFN-γ. TGFβ, IL-17, TNF-α, and MMPs are not ↑ (as in G5), NSS, and equal to G2/G3.	No
Morgan et al[45]	Rats (WI)	48	No	Ischemic: Left colon resection + Vessel ligation. End-to-end interrupted. Air checked	1 × 106 XENOG human ASCs	16 ASCs Gelatin sponge wrapping	Anastomosis. Anastomosis + gelatin sponge wrapping	Follow-up: Macroscopic. MBP in situ. Histology, IF, rtPCR. SC tracing	3 and 7 d	No mortality/complications. ASCs: ↓ AL and abscesses (3, 7 d); ↓ adhesions (3 d). No changes in MBP. ASCs ↑ collagen and microvascular density. Labelled cells in submucosa and muscularis. No SS differences in rtPCR.	No
Small bowel anastomoses
Maruya et al[46]	Pigs	7	Yes (anast)	High risk: vessel ligation + local mitomycin C. 8/animal. Multilayer end-to-end	AUT ASCs	28 anastomoses wrapped with 3 ASCs sheets	Anastomosis without sheets	MBP, histology and hydroxyproline (5, 7d). mRNA (1, 7d)	1, 5, 7 d	ASCs: MBP ↑ at 7 d, similar to normal healing. ASCs ↑ hydroxyproline at 7 d. ASCs ↑ submucosal collagen 7 d (??P). ASCs: ↑ FGF2, COL1A1 and COL3A1 day 1 and COL1A1 and COL3A1 day 7.	No
Pan et al[47]	Pigs	16	No	5/animal. Section. Functional end-to-end (energy sealing device)	0.5 × 106 ALLO ASCs	8 × 5 anastomoses. Local injection	Anastomosis without ASCs	Follow-up: Macroscopic. MBP. Histology, IHQ, IF, western, PCR arrays. SC tracing	7, 14 d	NSS in complications/leakage and MBP. ASCs: Reepithelialization and ↑ collagen at 7 d (??P). ASCs ↑ proliferation, and ↓ CDH1, SMAD3, STAT3, TGF-α, VEGFA. Labelled cells in mucosa.	1 death in ASCs (ileus)
Digestive (gastric) perforations
Komiyama et al[48]	Rats (WI)	40	No	Greater curvature incision. Block continuous suture	1 × 107 AUT ASCs	20 local injection	PBS local injection	Histology day 7 (n = 5), day 28 MBP, day 7 (n = 5) SC tracing	7 and 28 d	Labelled cells at 7, 28 d without differentiation. ASCs ↑ neovascularity and connective tissue at 7 d and ↓ connective tissue at 30 d. MBP ↑ 7 d with ASCs.	No
Liu et al[49]	Rats (SD)	108	No	2 cm body incision. Interrupted suture	5 × 106 AUT ASCs	24 local injection. 24 topical in fibrin glue	Sham operated. PBS injection. Topical fibrin glue	Macroscopic. Histology. IHQ, IF, western. SC tracing	3, 5, 7 d	Injected ASCs ↓ severe adhesions (3, 5, 7 d), dehiscence (3 d), abscesses (7 d). 20% total healing at 7 d (vs 0%). ASCs ↑ MBP (5, 7 d). Injection the highest values (comparable to sham operated at 5 d). ASCs ↓ inflammation and ↑ granulation (5, 7 d, ??P), more with injections. Injected ASCs ↓ IL-6 (day 5, 7) and ↑ TGFβ1 (day 3, 5). Label+ cells submucosa/granulation, differentiation+.	No
Tanaka et al[50]	Rats (SD)	30	N	5 mm incision. No suture	ALLO myoblasts sheet	15 sheet placed with shifter	No suture	Macroscopic (adhesion). Blood and ascites. Histology. SC tracing	3, 5, 10, 20 d	Sheets ↓ adhesions in all periods. Histology: sheets regenerated mucosa and muscle; control connective tissue (??P). Myoblast in gastric wall. ↓ SS peritoneal fluid hyaluronic acid (??P) all periods.	No
Oesophageal anastomotic leakage/fistula
Xue et al[51]	Rabbits	21	No	Transection, incomplete anast, tube during 7 d.	2 × 106 AUT MSCs	12 MSCs in fibrin sealant in fistula	9 fibrin sealant	Cervical MRI (5 wk). Macroscopic, histology, IF, cytokine at 8 wk. SC tracing	5, 8 wk	MRI: ↓ inflammatory reaction MSCs. Macroscopic: ↑ closure and ↓ infection MSCs. Histology/IF: MSCs survive & differentiate. Milder inflammation and less collagen (??P) with MSCs. MSCs: ↑ IL-10, MMP-9 and ↓ TNF-α, TGF-β.	5/9 control, 3/12 MSCs died (NSS)
Biliary anastomoses leakage/stenosis
Zhang et al[52]	Pigs	9	No	CBD transection. Running sutures	4 × 106 AUT ASCs	3/3 stent + mesh with ASCs. Topical ASCs	3 plastic stent + vycril mesh	Serum BQ (0, 7, 30 d). Cholangiogram 30 d. Histology, IHQ and IF 30 d	0, 7, 30 d	No clinical/laboratory suggesting cholestasis. No leaks/stenosis on cholangiogram (??P). Topical ASCs ↑ SS CD44, CD34 (MSCs) and CD31 (angiogenesis) and ↓ fibrosis and inflammation (??P).	1 death (ASCs + mesh) – cholangitis
Hara et al[53]	Pigs	11	No	Hepatic conduct section. End-to-end running (post)/interrupted (ant)	AUT ASCs	6 ASCs sheets around anastomosis	5 anastomosis without sheets	Blood (0, 7, 14 d). Macroscopic, histology at 14 d	0, 7, 14 d	No leakages, abscesses, mortality, lab cholestasis. Macroscopic: CBD diameter higher in controls due to wall thickening. Histology: ↓ inflammation, collagen and ↑ small vessels with ASCs (??P)	No

Only statistically significant or highly relevant results are shown, the last remarking their statistical value. SCs: Stem cells; MSCs: Mesenchymal stem cells; BM-MSCs: Bone marrow-derived mesenchymal stem cells; ASCs: Adipose-derived stem cells; IV: Intravenous; MBP: Medium bursting pressure; IHQ: Immunohistochemistry; IF: Immunofluorescence; PCR: Polymerase chain reaction; N: Number; anast: Anastomosis; Perf: Perforation; AUT: Autologous; ALLOG: Allogeneic; SYNG: Syngeneic; XENOG: Xenogeneic; SD: Sprague-Dawley; WI: Wistar; SSF: Saline solution; NSS: Non-statistically significant; SS: Statistically significant; ??P: No statistics provided; PLP: Polypropylene; CS: Culture solution.