Adipose Tissue Stromal Fraction Effect in the Treatment of Osteochondral Defects in Rabbits

doi:10.21203/rs.3.rs-3136616/v1

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Adipose Tissue Stromal Fraction Effect in the Treatment of Osteochondral Defects in Rabbits

https://doi.org/10.21203/rs.3.rs-3136616/v1

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Background:

This study aimed to evaluate the efficiency of stromal derived from adipose tissue in the treatment of osteochondral defects in rabbits. A total of 36 white New Zealand rabbits were used: 24 to perform an osteochondral defect in the trochlear groove of the joints and 12 to collect adipose tissue in the region of the adipose panicle, divided in three digestion times (30, 45 and 50 minutes). The groups were divided into negative control (C-), stromal fraction of adipose tissue (SFAT) and positive control (C+) with autogenous osteochondral graft.

Results:

Clinical, radiographic, macroscopic and histopathological evaluations showed that the therapeutic effects of the stromal fraction of adipose tissue minimized the degenerative signs of articular cartilage in the treatment of osteochondral defects in rabbits.

Conclusion:

The digestion time of 30 minutes was the best option for obtaining the stromal fraction from adipose tissue of rabbits and its use at the lesion site was satisfactory, without any immunological complications, indicating potential as an alternative for the treatment of osteochondral defects.

Rabbit

mesenchymal cells

joint

adipose tissue stromal

One of the great challenges in veterinary and human orthopedics is the treatment of osteoarticular lesions. The biggest complication of joint injuries is the development of degenerative joint disease (DJD), characterized by being irreversible, progressive and potentially debilitating, and the most frequent joint alteration in both humans and companion animals [1, 2].

Stem cells (SCs) are one of the world's hopes for the treatment of diseases in which conventional therapies are not efficient and can be found in all types of tissue in greater or lesser amounts [3, 4]. Mesenchymal stem cells (MSCs) are undifferentiated cells that, if properly stimulated, have a high capacity to multiply and differentiate into other cell types, such as osteocytes, chondrocytes, tenocytes, myocytes and adipocytes [5]. They also have high self-renewal potential, which holds promise for the treatment of degenerative diseases [6, 7, 8, 9]. Thus, the tissues most used as a source of MSCs are bone marrow and adipose tissue [10, 11].

MSCs derived from adipose tissue (ASCs) have advantages due to ease of collection and isolation, in addition to not being influenced by age [12], coexisting diseases [13] and other external factors such as the use of drugs [14]. With the possibility of differentiating ASCs in injured tissue, added to an immunomodulatory effect and easy extraction, the use of ASCs becomes an attractive alternative in the treatment of DJDs [15, 16, 17].

The aim of this study, therefore, was to evaluate the efficacy of adipose tissue-derived stromal fraction in the treatment of osteochondral defects in rabbits.

Radiographic evaluation showed progressive filling of defects over time in all study groups. The group treated with stromal fraction of adipose tissue (SFAT) showed greater area of new bone formation than the untreated group (C-) at 60, 75 and 90 days post-operative. The positive control showed a greater area of new bone formation in relation to the C- group at all evaluation times and did not differ from the SFAT group at 45, 60, 75 and 90 days post-operative (Table 2).

Table 1

Scoring system for macroscopic evaluation of the joint submitted to osteochondral grafting.

Adapted from [18].
Category	Score	Category	Score
1. Contracture		6. Reparis tissue color
No	0	Normal	0
Yes	1	Translucent	1
2. Effusion		Dark	2
No	0	7. Tissue repais superficie
Yes	1	Smooth	0
3. Intra-articular adherence		Fibrillated	1
No	0	Degenerated	2
Yes	1	8. Integration with adjacente cartilage
4. Synovitis		Complete integration	0
No	0	Demarcated edges	1
Yes	1	9.Defect filling
5. Osteophytes		Leveled with adjacente cartilagem (total)	0
No	0	50% of defect depth repair (partial)	1
Yes	1	0% of defect depth repair	2

Table 2

Mean and standard deviation of bone neoformation area obtained by radiographic evaluation performed over 90 days of osteochondral defects created in the femorotibiopatellar joint, untreated (C-) or treated with stromal fraction of adipose tissue (SFAT) or autogenous osteochondral graft (C+).
Avaliation time	15 days	30 days	45 days	60 days	75 days	90 days
C-	1,58 (1,34) ^a	3,21(1,85) ^a	4,57(1,70) ^a	5,2(0,84) ^a	5,8(1,10) ^a	8,0(1,58) ^a
SFAT	1,67(2,30) ^a	3,5(1,00) ^a	5,50(2,36) ^ab	6,8(0,84) ^b	7,8(1,30) ^b	9,8(0,84) ^b
C+	3,93 (1,50) ^b	5,29(2,70) ^b	6,46(1,81) ^b	7,6(1,52) ^b	8,8(1,30) ^b	11,6(0,74) ^b

Means followed by different lowercase letters in the columns differ from each other (p < 0.05) by the SNK test.

The macroscopic evaluation performed 45 days after surgery showed lower intra-articular adhesions in the SFAT group compared to the C + and C- groups. At 90 days of evaluation, the C- group remained with greater adherence in relation to the C + and SFAT groups, which did not differ from each other. No significant differences in synovitis scores were observed among the groups at 45 days, but at 90 days the SFAT and C + groups showed similar and significantly better synovitis scores than the C- group. No difference was observed in the repair tissue staining score among all groups at 45 days. At 90 days, the SFAT group had a significantly lower repaired tissue staining score than the C- group, but higher than the C + group. Repair tissue surface scores were similar between the SFAT and C + groups, and significantly better than the C- group at all assessment times. Level filling and complete integration with adjacent cartilage were observed in defects in all groups, both at 45 and 90 days of evaluation (Table 3).

Table 3

Mean and standard deviation of parameter scores for macroscopic evaluation of the femorotibiopatellar joint in rabbits with osteochondral defects, untreated (C-) or treated with stromal adipose tissue fraction (FETA) or autogenous osteochondral graft (C+) evaluation performed over 45 and 90 days.
Category	C-	SFAT	C+	C-	SFAT	C+
Category	45 days			90 days
Contracture	0,33(0,50)^a	0,00(0,00)^a	0,00(0,00)^a	0,25(0,46)	0,17(0,41)	0,13(0,35)
Effusion	0,00(0,00)^a	0,00(0,00)^a	0,00(0,00)^a	0,00(0,00)^a	0,00(0,00)^a	0,00(0,00)^a
Adherence	1,00(0,00)^a	0,16(0,41)^b	1,00(0,00)^a	1,00(0,00)^a	0,00(0,00)^b	0,13(0,35)^b
Synovitis	0,33(0,50)^a	0,00(0,00)^a	0,11(0,33)^a	0,75(0,46)^a	0,00(0,00)^b	0,00(0,00)^b
Osteophytes	0,89(0,33)^a	0,50(0,55)^a	0,89(0,33)^a	0,88(0,35)^a	0,33(0,52)^a	0,50(0,53)^a
Color	1,22(0,97)^a	1,00(0,63)^a	0,78(0,97)^a	1,50(0,93)^a	0,50(0,55)^ab	0,38(0,74)^b
Superficie	1,33(0,71)^a	0,00(0,00)^ab	0,56(0,88)^b	1,75(0,46)^a	0,00(0,00)^b	0,38(0,74)^b
Integration	0,89(0,33)^a	0,83(0,41)^a	0,78(0,44)^a	0,88(0,35)^a	0,50(0,55)^a	0,50(0,53)^a
Filling	0,89(0,60)^a	0,83(0,41)^a	0,89(0,60)^a	0,88(0,35)^a	0,83(0,41)^a	0,63(0,52)^a
Total	6,75(2,71)^a	3,33(1,36)^a	4,44(2,69)^a	7,87(1,64)^a	2,33(1,03)^b	2,62(1,99)^b

Means followed by different lowercase letters in the lines differ from each other (p < 0.05) by the Krukal Wallis test followed by the Dunn test.

The histological evaluation of the negative control group (C-) showed filling of the periphery of the defect with spongy bone tissue composed of thin trabeculae and, of the central region, by fibrous connective tissue, in 100% (6/6) of the animals at both evaluation times. The defect surface was filled with fibrous connective tissue in 33.33% (2/6) of the animals at 45 days and 50% (3/6) of the animals at 90 days; or by fibrocartilaginous tissue in 66.66% (4/6) of the animals at 45 days and 50% (3/6) of the animals at 90 days (Fig. 3 and Fig. 4).

In the histological evaluation performed at 45 days, 50% (3/6) of the animals treated with stromal fraction of adipose tissue (SFAT) showed filling of the periphery of the defect with spongy bone tissue composed of trabeculae of intermediate thickness and, of the central region, by fibrous connective tissue and intense neovascularization. In the other 50% (3/6) the periphery of the defect was filled with spongy bone tissue composed of thick trabeculae and the central region with fibrocartilaginous tissue. The surface of the defect was filled by fibrous connective tissue in 50% (3/6) of the animals and by fibrocartilaginous tissue in 50% (3/6) of the animals at 45 days. At 90 days, the defect was completely filled with spongy bone tissue composed of thick trabeculae in 100% (6/6) of the animals. The defect surface was filled by fibrocartilaginous connective tissue in 50% (3/6) of the animals and by cartilaginous tissue in 50% (3/6) of the animals; however, the surface was irregular and with some cracked areas (Fig. 3 and Fig. 4).

The histological evaluation of the positive control group (C+) showed complete filling of the defect with spongy bone tissue composed of thick trabeculae in 100% (6/6) of the animals, at both evaluation times, with small areas of endochondral ossification being observed at 45 days. The defect surface was filled with fibrocartilaginous tissue in 100% (6/6) of the animals. At 90 days, filling of the surface of the defect by fibrocartilaginous tissue was observed in 16.66% (1/6) of the animals and by cartilaginous tissue in 83.33% (5/6) of the animals (Fig. 3 and Fig. 4).

Radiographic, macroscopic and histological evaluations showed a similar pattern of bone repair between the SFAT and C + groups, with gradual filling of the defect from the periphery to the center of the lesion with spongy bone composed predominantly of thick trabeculae and active osteoblasts in widened gaps. On the other hand, bone repair in group C- occurred more slowly and with a predominance of thin trabeculae in the periphery of the lesion and fibrous tissue in the center of the lesion. The repair of the chondral lesion was also similar between the SFAT and C + groups, showing repair with a predominance of fibrocartilaginous and cartilaginous tissue while in the C- group there was a predominance of fibrous tissue on the joint surface. Any therapy that shows similar or equal results to osteochondral autograft can be considered an efficient treatment with great potential for practical use in the treatment of joint diseases [18].

Although studies have shown beneficial therapeutic effects of the use of mesenchymal stem cells in the treatment of osteochondral lesions [20, 21] this is still an expensive technique that requires infrastructure for isolation and cell culture, which the makes it unavailable to most patients. The use of the stromal fraction of adipose tissue as a source of MSCs allows the treatment of osteochondral lesions with minimal laboratory infrastructure and at a low cost, allowing the immediate treatment of a greater number of patients.

Studies have already shown that AD-MSCs have immunomodulatory and anti-inflammatory effects and are able to decrease degenerative signs if injected into human osteoarthritic joints [22, 23]. These same studies showed that the effect is dose dependent, and the greater the amount of AD-MSCs injected into the joint, the better the results.

Obtaining adipose tissue from the interscapular region in the adipose panicle was easy to perform and provided a sufficient amount of tissue for the production of the stromal fraction of adipose tissue (SFAT), in addition to not developing postoperative complications. In his review, [24] had already shown several studies that validate the effectiveness of the stromal fraction production technique and the collection site, but it was not clear whether the amount of adipose tissue in the panicle would be sufficient for the production of SFAT of therapeutic use without the need for cultivation and expansion. Other authors, such as [8, 23], chose to obtain MSCs from bone marrow aspiration, a technique that has the advantage of being simpler; however, it has disadvantages such as the drastic reduction in the number of cells with advancing age [12]. In addition, bone marrow MSCs are more influenced by external factors such as disease [13] and drug use [14]. [10, 11], observed better results with the technique of extracting MSCs in the gluteal subcutaneous region (adipose tissue) in dogs compared to extraction in the bone marrow of the tibial plateau.

The stromal fraction of adipose tissue has different cell types, such as MSCs, fibroblasts and endothelial cells. The cells of greatest therapeutic interest are MSCs, which have a great ability to differentiate into different cell lines [8, 9, 10, 11, 12] justifying the choice of stromal fraction in the treatment of osteochondral defects.

The present protocol has advantages over the techniques of MSC isolation from adipose tissue, as it is easy to perform in environments without much equipment or materials. In addition, it allows the immediate use of cells for the early treatment of diseases and, consequently, a better therapeutic result. It has already been described that MSCs can exert their effect through the secretion of cytokines and other molecules, for paracrine effect. It is known that the secretion profile of MSCs is altered by the microenvironment in which they are placed, so that MSCs exposed to environments rich in pro-inflammatory mediators, develop an anti-inflammatory secretion profile. In contrast, if MSCs were exposed to a microenvironment rich in cytokines and other anti-inflammatory molecules, they acquire a predominantly pro-inflammatory secretion profile. Early treatment increases the chances of MSC exposure to a pro-inflammatory environment, improving the therapeutic outcome [15, 16, 17].

Based on the results obtained in the present study, we can conclude that SFAT is an alternative for the treatment of osteochondral defects and has a therapeutic result similar to that of osteochondral autograft, in addition to being easily obtained and applied to the lesion site without any immunological complications, for be autologous cells.

Animals

The research project was approved by the Comitê de Ética em Experimentação Animal of the Universidade de Uberaba (n. 033/2014). Thirty-six male New Zealand rabbits (Oryctolagus cuniculus), aged 3 to 4 months, with an average body mass of 2.5 kg, were used.

Evaluation of the stromal fraction of adipose tissue in the treatment of osteochondral defects employed 24 rabbits, totaling 48 femorotibiopatelar joints (FTPs). The right joints were randomly divided into two experimental groups with 12 joints each. One group was treated with stromal fraction of adipose tissue (SFAT) and the other with autogenous osteochondral graft, constituting the positive control (C+). The osteochondral defects of the left FTP joints were not treated, constituting the negative control (C-). An additional 12 rabbits were used to obtain the stromal fraction of adipose tissue.

Acquisition of stromal fraction of adipose tissue in rabbits.

Samples collected from adipose tissue were placed in a 50-mL sterile Falcon tube containing 25 mL of 0.9% saline solution and immediately sent to the Laboratório de Pesquisa em Sanidade e Produção Animal nos Trópicos for processing. All sample manipulation was performed aseptically in a laminar flow hood. Initially, the samples were shaken to remove blood and cellular debris and transferred to a new 50-mL Falcon tube containing 20 mL of 0.1% collagenase type I solution (Roche®, Germany) diluted in 0.015 M PBS. The tissue was fragmented with the aid of scissors, generating particles of about 3 mm. The samples were then divided into three digestion times, remaining in an oven at 37.5°C for 30, 40 and 50 minutes with vigorous shaking at 5-minute intervals. After digestion, the samples were centrifuged for 10 minutes at 1400rpm, the supernatant discarded and the pellet containing the stromal fraction resuspended in 20 mL of 0.015 M PBS for washing and removal of collagenase residues. The sample was then centrifuged for 10 minutes at 1400rpm and the supernatant discarded. The pellet was evaluated for cell viability by Trypan blue.

Creation of osteochondral defects and treatments

Surgeries were performed in the operating room of the Hospital Veterinário de Uberaba. The animals were anesthetized with 5mg/kg xylazine, IM (Xilazin®, Syntec, Brazil) and 40mg/kg ketamine, IM (Cetamin®, Syntec, Brazil), followed by lumbosacral epidural block with 0.5% 0.22mL/Kg bupivacaine (Neocaína®, Cristália, Brazil) and 0.1mg/kg Morphine (Dimorf®, Cristália, Brazil). The following were then administered: 40 mg/kg ceftriaxone sodium, IM (Eurofarma, Brazil) and 0.1mg/kg meloxicam, IM (Maxicam® 0.2%, Ouro Fino, Brazil).

Craniolateral access was performed to the right femorotibiopatellar joint and, moving the patella away medially, the middle third of the trochlear sulcus was located, the site chosen for creating the osteochondral defect [18]. With the aid of a drill and a perforation limiter, a hole measuring 5 mm in diameter and 5 mm in depth was made in the chosen location (Fig. 1). The defects (osteochondral holes) of the right joints of the animals in the SFAT and C + groups were filled with, respectively, 0.3mL of the stromal fraction of adipose tissue (SFAT) (Fig. 1) and a macerated osteochondral graft, which was removed when the drill was inserted to make the hole (osteochondral defect) in the trochlear groove. Left joint defects were not filled, constituting the negative control. After surgery, animals received 5mg/kg morphine, SC, every 6 hours for the first 24 hours. On the second and third days after surgery, the animals received 5 mg/kg tramadol, SC (Tramadon, Cristalia, Brazil), every 12 hours.

Radiographic evaluation

Radiographic evaluations were performed in the immediate postoperative period and at 15, 30, 45 days for all animals and at 60, 75 and 90 days for half of the animals in each group. Two radiographic exposures were performed in the mediolateral and craniocaudal projections. The area of radiolucency (unfilled) in the osteochondral defect was measured and the area of new bone formation was obtained as the difference between the radiolucency area in the post-immediate period minus the radiolucency area of each evaluation time, using the program ImageJ 1.52k (US National Institutes of Health, Bethesda, Maryland, USA).

Macroscopic and histopathological evaluation

Macroscopic evaluation of the femorotibiopatellar joints was performed after euthanasia, through photographic registration and use of the inverse scoring system adapted by [18] (Table 1). Six animals from each group were evaluated at 45 days, and another six at 90 days, after surgery.

The scores used for the macroscopic evaluation were based on the following criteria: contractures; effusions; intra-articular adhesions; synovitis; osteophytes; repair tissue staining; repair fabric surface; integration with adjacent cartilage and filling of the defect.

Microscopic evaluations were performed with six joints from each group at 45 days, and another six at 90 days, after surgery. These assessments were performed using only the joint region. After collection, joints were fixed in 10% buffered formalin. The joints were subsequently submitted to demineralization with an aqueous solution of 10% v/v formic acid and 9% v/v sodium citrate for 180 days. After this period, the bones were sectioned transversally and the halves processed by routine embedding in paraffin, stained with hematoxylin-eosin and descriptively evaluated by optical microscopy. The type and characteristic of the filling tissue observed in the periphery, center and surface of the osteochondral defect were evaluated (Fig. 2).

Statistical evaluation

A completely randomized design was used. Parametric data (cell count and area of new bone formation) were submitted to analysis of variance (ANOVA) and means of cell counts were compared by the paired t test and those of area of new bone formation by the unpaired t test. Nonparametric data (scores) were submitted to the Kruskal-Wallis test followed by the Dunn test using the program Graphpad Instat 3 (GraphPad Software Inc., United States). Differences were considered significant if p < 0.05 [19].

Acknowledgements

UNIUBE – University of Uberaba for space and laboratories

Authors’ contributions

A.B.O.; M.C.M.; I.R.R.; I.M.; B.M.B.; S.A.O.M.; F.C.D.M.; E.G.L.A. was involved in conception, article design and interpretation of data and revising the article critically for important intellectual content, and gave final approval of the version to be published. All authors have read and agreed to the final version of the manuscript.

Data Availability

All data generated or analysed during this study are included in this published article.

Ethics approval and consent to participate

The experimental procedure was performed in accordance with the Regulations of Experimental Animal Administration rules of Laboratory Animal Center of UNIUBE and with the ARRIVE guidelines. The experiment was approved by the Comitê de Ética em Experimentação Animal of the Universidade de Uberaba (n. 033/2014).

Consent for publication

Not applicable.

Competing interests

The author reports no conflicts of interest in this work.

Funding

Not applicable.

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No competing interests reported.

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Adipose Tissue Stromal Fraction Effect in the Treatment of Osteochondral Defects in Rabbits

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Abstract

Figures

Introduction

Results

Discussion

Conclusion

Materials and Methods

Animals

Creation of osteochondral defects and treatments

Radiographic evaluation

Macroscopic and histopathological evaluation

Statistical evaluation

Declarations

References

Additional Declarations

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