Adverse effects of stromal vascular fraction during regenerative treatment of the intervertebral disc: observations in a goat model

A total of twelve female adult Dutch milk goats underwent a surgical procedure to induce disc degeneration by injecting five lumbar discs through a left retroperitoneal approach with 0.25 U/ml Chondroitinase ABC (CABC; Sigma, St. Louis, MO, USA) as described before [8]. Disc degeneration was allowed to develop for 3 months, after which this study was designed as a time-oriented study. During the second surgical procedure, ~100 g of perirenal adipose tissue was harvested and SVF was isolated as described by Zuk et al. [16] with minor modifications. The obtained adipose tissue was washed with phosphate-buffered saline (PBS) to remove the majority of red blood cells, chopped into small pieces of about 50 mm³, and the extracellular matrix was digested for 60 min at 37 °C with 1 U of collagenase (“Blendzyme”, Roche Diagnostics, Brussels, Belgium) per gram adipose tissue in PBS. A single cell suspension was obtained by filtering the digested material through a 100-μm mesh filter (Stokvis and Smith B.V., IJmuiden, The Netherlands) to remove tissue debris. The adipose stem cell-containing cell suspension was centrifuged at 600g for 10 min, and the pellet was resuspended in culture medium, which was composed of Dulbecco’s modified eagle medium (DMEM, Gibco, Paisley, UK), supplemented with 500 µg/ml streptomycin sulphate (Sigma), 600 µg/ml penicillin (Sigma), 50 µg/ml gentamicin (Gibco), 2.5 µg/ml fungizone (Gibco), and 10 % foetal bovine serum (FBS, Hyclone, Logan, UT). After centrifugation, the SVF pellet was resuspended in DMEM without additives, washed for an additional two times with PBS, and subsequently resuspended to either 10⁶ or 10⁷ cells/ml PBS. In each goat, four degenerated discs were injected with 100 µl containing either 10⁵ or 10⁶ freshly isolated nucleated cells or plain PBS as a control treatment, in a randomised fashion, using a 26-Gauge needle. After wound closure, the animals were allowed to move freely with follow-up periods of 1 (n = 7) and 3 months (n = 5). Prior to killing, goats were sedated with 10 mg/kg ketamine intramuscularly, and lateral radiographs were obtained. Subsequently, they were euthanised with an intravenous overdose of sodium pentobarbital (20 mg/kg), after which the lumbar spines were harvested, dissected and prepared for further analysis.

Histologically, the intervertebral discs used as sham controls had a normal appearance with concentric annular lamellae, which were easily distinguishable from the nucleus pulposus tissue (Fig. 2a). The extracellular matrix of the latter had an open structure with chondrocyte-like cells present in the matrix. The enzymatically degenerated discs injected with PBS in the second surgery (sham treatment) had a more degenerated appearance (Fig. 2b). In these discs, annular fissures were observed, as well as a condensation of the extracellular matrix of the nucleus pulposus with a decreased number of cells and a diminished demarcation between annulus fibrosus and nucleus pulposus (Fig. 2b). In 38 of 48 of the discs injected with SVF fraction, a severe inflammatory response occurred (Fig. 2c). Ten out of 14 discs injected with 10⁵ perirenal SVF demonstrated an inflammatory response and the same was observed in 13/14 discs injected with 10⁶ cells after 1 month. After three months, this response was observed in 7/10 and 8/10, respectively. In the central region a cellular infiltrate, mainly consisting of plasma cells and macrophages, was observed in the early phase (1 month) of the response (Fig. 2d). At the margins, neovascularisation was observed in the disc and osteoclastic activity was high at the bone–infiltrate junction (Fig. 2e). After three months, (myo)fibroblastic cells were observed in the central area where extensive remodelling (scar tissue formation/collagen deposition) occurred. This reaction was similar to the response that was observed in discs injured with higher doses of Chondroitinase ABC [8]. Also, osteoblasts were found at the margins rimming along osteoid. Simultaneously, we conducted a similar pilot study, using hyaluronic acid as a vehicle, in which we observed the same response and adverse effects (data not shown).

After this first regeneration study using autologous SVF in a goat model, two hypotheses were formulated as potential causes of the observed inflammatory response. First, residual enzyme activity of Chondroitinase ABC was suggested as a possible culprit for the adverse effects. Second, the reaction might have stemmed from the presence of macrophages in combination with red blood cells (RBCs) in the cell mixture. After all, RBCs had previously been reported to induce cartilage damage in the joints of haemophilia patients, resulting in haemophilic arthropathy by the iron-catalysed production of destructive oxygen metabolites [17]. To determine the trigger for the inflammatory response in our first study, an additional study was designed, in which the goal was threefold: (1) to rule out that adverse effects had been due to residual CABC activity, (2) to exclude the large number of RBCs as causing mechanism and (3) to investigate a more homogeneous stem cell fraction. For the first purpose, SVF was also injected into healthy goat discs; for the second goal, SVF was treated with a density gradient (Optiprep) to get rid of the RBCs; and third, adipose-derived stem cells (ASCs) were cultured before implantation in goat IVDs.

To rule out that adverse effects were due to residual CABC activity (after 3 months incubation time to develop degeneration), one healthy disc in each of six goats was injected with SVF. Furthermore, plain SVF, density gradient-treated SVF and cultured ASCs were randomly evaluated in four chemically degenerated discs, using the same goat intervertebral disc degeneration model as in the first study. For the two SVF groups (plain SVF and density gradient-treated SVF), the same protocol was followed in this second study for the harvesting of SVF. After re-suspension in DMEM without additives, the SVF was divided into two equal parts. One part was further processed as described for the first study (plain autologous SVF), whereas the other part was subjected to Optiprep^® density gradient centrifugation. Optiprep^® density gradient-mediated SVF purification through reduction of red blood cells (RBCs) was performed according to the manufacturer’s instructions (protocol C2, Axis-shield). The mononuclear stromal vascular fraction band was collected, washed twice with PBS, and finally resuspended to 10⁶ nucleated cells/ml in PBS. Of this cell suspension, 100 µl was injected in the enzymatically degenerated discs. For the cultured ASCs group, adipose tissue had already been harvested during the degeneration induction surgery, and these cells were resuspended in culture medium and seeded in 75 cm² culture flasks (Greiner Bio-One, Kremsmuenster, Austria). Upon confluency, adipose-derived stem cells (ASCs) were trypsinised from the culture flasks using 0.25 % trypsin/0.1 % EDTA in PBS, harvested, and reseeded once (passage 1). After harvesting, cells were stored in liquid nitrogen until one week before reinsertion. At that time, cells were thawed, allowed to recover for one week, and finally resuspended to 10⁶ nucleated cells/ml PBS just prior to implantation in the goat discs.

Both the healthy and the previously degenerated discs injected with plain SVF exhibited an inflammatory response similar to those described in the first study. We observed scar tissue formation in the central part of the nucleus pulposus and bone formation at the rims of the vertebrae. Discs injected with RBC-depleted SVF (Optiprep) demonstrated mild degeneration, although one disc out of six revealed similar endplate irregularities and an inflammatory response as described in the plain SVF-injected discs. CABC-degenerated discs injected with PBS showed identical results to discs injected with cultured ASCs, i.e. no inflammatory response or endplate irregularities in any disc, thus ASCs themselves showed no adverse effects at all. A regenerative response could not be identified in these discs either, although some discs did show increased numbers of nucleus pulposus-like cells [18].

This second study attempted to pinpoint the cause of the adverse events by applying (a) density gradient removal of RBCs; (b) ASCs cultured to homogeneity (to remove RBCs as well as other contaminating cell types present in SVF); and (c) injection of SVF in intact discs to rule out CABC-mediated effects. It was concluded that RBCs indeed might have been the cause of the adverse effects and that the adverse effects were not due to the CABC that was used to create the degenerative disc model. Furthermore, we concluded that cultured ASCs did not regenerate the disc after mild degeneration, but neither did this treatment lead to adverse effects. In contrast, a similar experiment had been performed in a canine model, in which none of these adverse events were described [19]. This led us to the formulation of several other potential causes for the adverse events observed in our first two studies, based on the differences between our studies and the dog study, namely (a) residual collagenase activities in the SVF mixture; (b) the enzymatically induced disc degeneration, in contrast with the herniation model used in the referred study; (c) specific interaction based on the goat species used; and (d) potential cell mixture differences due to the harvest location of the adipose tissue. To investigate these newly formulated hypotheses, a third goat study was conducted.

During the first surgical procedure, the nucleus pulposus from three (n = 2 goats) or four (n = 2 goats) disc levels (randomly between T13–L1 and L5–L6) were partially excised under open surgical dissection. The annulus fibrosus was allowed to heal for approximately six weeks prior to disc therapy. In the second procedure, adipose tissue was harvested from both the perirenal area and a subcutaneous site, and transferred to a cell processor. Adipose tissue was weighed, transferred to tissue culture grade plastic dishes, and minced finely with scissors and/or scalpels under sterile conditions. The minced tissue was then washed with warmed Lactated Ringers, after which 1 U/ml collagenase (CELASE™, Cytori Therapeutics Inc, San Diego, CA, USA) was added to the tissue. After approximately 50 min of digestion, the digested tissue was further washed and concentrated, by spinning. One additional washing step was added in comparison with the protocol of the first two studies to further minimise residual enzyme activity. Under fluoroscopic visualisation, the appropriate test material was applied through a right-lateral approach by injection with a 25-Gauge needle into the appropriate level as described before. The nucleotomised IVDs received 100–200 µl of hyaluronic acid vehicle mixed with either 2 × 10⁶ nucleated cells/ml isolated from perirenal fat (perirenal SVF), 2 × 10⁶ nucleated cells/ml isolated from subcutaneous fat (subcutaneous SVF) or hyaluronic acid vehicle with PBS (sham control) in a randomised fashion. The intact discs received 0.1 ml of 10⁷ nucleated cells/ml isolated from perirenal fat, 10⁷ nucleated cells/ml isolated from subcutaneous fat or PBS in a randomised fashion. In total, 24 discs were involved in the experiment; the follow-up period was four weeks. In parallel to the animal study, characterisation of SVF from the different sources was performed including red blood cell counts, determination of ASC colony frequency and analysis of residual enzyme activity in the last PBS washing step, using caseinolytic and gelatinolytic assays.

The histological results of this third study were promising as the discs looked largely unaffected by the injection of different treatment groups. Annulus layers were still outwardly folded, no inflammatory responses, no increased cellularity, and no apparent differences between the groups were observed. Red blood cell numbers were 10⁸–10⁹ cells per ml SVF and residual enzyme levels were negligibly low or even below assay baseline threshold.

After the successful elimination of adverse events in the third goat study, we concluded that adding an extra washing step to the SVF harvesting protocol had eliminated the adverse events. Furthermore, subcutaneous adipose tissue appeared to yield a higher number of ASCs per ml SVF obtained and showed similar values as described for human SVF. Therefore, in the next study design for disc regeneration, the subcutaneous harvest location was used.

This study was performed in the context of a disc regeneration study using a newly developed hyaluronic acid-fibrinogen hydrogel (ratio 1:18 w/w, MW 235 kDa, ProCore Bio Med Ltd, Ness Ziona, Israel) and BMP-2 (200 ng/ml, UHZ, Zürich, Switzerland). In seven adult Dutch milk goats, five lumbar IVDs were chemically degenerated during 3 months using 0.25 U/ml Chondroitinase ABC. During the second procedure, subcutaneous adipose tissue was harvested and SVF was isolated, following the same protocol as described before in the previous studies. Subsequently IVDs were randomised to five intervention groups: hyaluronic acid-fibrinogen vehicle only, vehicle with SVF (10⁶ nucleated cells), vehicle with BMP-2, vehicle with both SVF and BMP-2 or no intervention. After killing at 3 months follow-up, lumbar spines were harvested for further analysis, using lateral radiographs, T2-weighted MRI, macroscopic and histological scoring.

This fourth study demonstrated once more an inflammatory response in 83 % of the discs injected with autologous SVF, concurrent with severe degenerative changes in all parameters. Figure 3 shows typical examples of osteophyte formation (3a) and endplate destruction (3b) on lumbar radiographs, as well as a complete loss of morphological organisation on T2-weighted MRI (3c). Nota bene: on the MRI images, the apophyses adjacent to the endplates are still visible, as in adult goats the growth plates persist after skeletal maturity [20]. No adverse effects were observed after injection of the hydrogel alone or in combination with BMP-2 into the mildly degenerated IVDs. In particular, no osteophytes were present in the group injected with vehicle and BMP-2 only.

Macroscopically, massive scarring was observed in the SVF-injected IVDs, including endplate destruction and osteophyte formation (Fig. 4). On histological sections, scar formation, endplate destruction and inflammatory cells, including macrophages and plasma cells were observed, indicating a chronic inflammatory response. Also, neovascularisation was observed in the discs as well as osteoclasts at the interface between NP and endplates. We saw no signs of infection, such as microorganisms, neutrophil or eosinophil granulocytes. Healthy discs were characterised by a well-defined demarcation between the NP and AF, organised collagen fibres and a normal gelatinous appearance of the NP matrix.