In silico modeling the potential clinical effect of growth factor treatment on the metabolism of human nucleus pulposus cells

Abstract

Background: While growth factors have the potential to halt degeneration and decrease inflammation in animal models, the literature investigating the effect of dosage on human cells is lacking. Moreover, despite the completion of clinical trials using growth differentiation factor-5 (GDF-5), no results have been publicly released.

Aims: The overall objective was to quantitatively assess the effect of three clinically relevant concentrations of GDF-5 (0.25, 1, and 2 mg) as a therapeutic for disc regeneration.

Materials and methods: Firstly, this work experimentally determined the effects of GDF-5 concentration on the metabolic and matrix synthesis rates of human nucleus pulposus (NP) cells. Secondly, in silico modeling was employed to predict the subsequent regenerative effect of different GDF-5 treatments (± cells).

Results: This study suggests a trend of increased matrix synthesis with 0.25 and 1 mg of GDF-5. However, 2 mg of GDF-5 significantly upregulates oxygen consumption. Despite this, in silico models highlight the potential of growth factors in promoting matrix synthesis compared to cell-only treatments, without significantly perturbing the nutrient microenvironment.

Discussion: This work elucidates the potential of GDF-5 on human NP cells. Although the results did not reveal statistical differences across all doses, the variability and response among donors is an interesting finding. It highlights the complexity of human response to biological treatments and reinforces the need for further human research and personalized approaches. Furthermore, this study raises a crucial question about whether these potential biologics are more regenerative in nature or better suited as prophylactic therapies for younger patient groups.

Conclusion: Biological agents exhibit unique characteristics and features, demanding tailored development strategies and individualized assessments rather than a one-size-fits-all approach. Therefore, the journey to realizing the full potential of biological therapies is long and costly. Nonetheless, it holds the promise of revolutionizing spinal healthcare and improving the quality of life for patients suffering from discogenic back pain.

Keywords: GDF5; growth factors; in silico; intervertebral disc; metabolism; regeneration.

FIGURE 1

(A) Human nucleus pulposus (NP) cells were isolated from four patients undergoing discectomy procedures. Following monolayer expansion, cell spheroids were formed in 96‐well plates coated with a thin layer of 2% agarose to prevent attachment. (B) Cell spheroids were cultured in standard expansion media (XPAN) for 1 week prior to 2 weeks under growth differentiation factor‐5 (GDF‐5) stimulation. (C) After GDF‐5 stimulation, individual spheroids underwent metabolic analysis using a Seahorse XFe96 analyzer and biochemical analysis. (D) Metabolic rates for each experimental group were then computed in silico to predict the donor‐specific reparative effects of GDF‐5 on extracellular matrix and subsequent impact on the nutrient microenvironment.

FIGURE 2

(A) Spheroid viability was assessed across the experimental groups using live/dead staining to ensure viability remained high in each group prior to performing metabolic rate measurements. (B) Oxygen consumption rates (OCR, nmol/million cells/h) and (C) lactate production rates (LPR, nmol/million cells/h) for NP cell spheroids in either XPAN media as an untreated control (ctr) or stimulated with three different concentrations of growth differentiation factor‐5 (GDF‐5) (0.25, 1, and 2 mg) (N = 4). While there is an apparent trend of increasing OCR and LPR with GDF‐5 concentration, only the 2 mg group had a significantly higher OCR compared to the ctr (p = 0.045).

FIGURE 3

(A) Glycosaminoglycan (GAG) production rates and (B) collagen production rates for cell spheroids in either XPAN media (control [ctr]) or stimulated with three different concentrations of growth differentiation factor‐5 (GDF‐5) (0.25, 1, and 2 mg) (N = 4). While no statistically significant difference was detected between experimental groups, donor‐specific colors are used to highlight donor variability and trends in donor‐specific response across both GAG and collagen. (C) Corresponding histological evaluation for Donor 2 using hematoxylin and eosin (H&E) to stain for cells, alcian blue (AB) to stain for GAG and picrosirius red (PSR) to stain for collagen. Scale bar is 200 μm.

FIGURE 4

(A) Predicted donor‐specific glycosaminoglycan (GAG) matrix content in the nucleus pulposus (NP) of a Grade III intervertebral disc over 10 years following no treatment (control [ctr]) or an injection of 0.25, 1, or 2 mg of growth differentiation factor‐5 [GDF‐5]. (B) Percent (%) difference in GAG matrix within the NP, compared to the untreated ctr, 5 years after treatment with either 0.25, 1, or 2 mg of GDF‐5. Highlighting both the donor‐specific overall response to GDF‐5 injection and the donor‐specific response to dose concentration. Data colors refer to simulated treatment concentrations, not donor.

FIGURE 5

(A) Predicted donor‐specific central metabolite (glucose, oxygen, and pH) concentrations within the nucleus pulposus of a Grade III intervertebral disc (N = 4) either untreated or subjected to growth differentiation factor‐5 (GDF‐5) injection (0.25, 1, and 2 mg). (B) Predicted glucose, oxygen, and pH distribution across a quadrant of a human intervertebral disc (Donor 4) with no treatment (control [ctr]) or with an injection of 0.25, 1, or 2 mg of GDF‐5. (C) Percent (%) reduction in central metabolite (glucose, oxygen, and pH) concentration relative to the donor‐specific ctr (N = 4).

FIGURE 6

(A) Predicted average (N = 4) glycosaminoglycan (GAG) matrix regeneration in the nucleus pulposus (NP) of a Grade III intervertebral disc over 10 years following no treatment (control [ctr]), an injection of unstimulated discogenic cells (1, 5, or 10 million), a growth differentiation factor‐5 (GDF‐5)‐only injection (1 mg), or GDF‐5 stimulated (1 mg) discogenic cells (1, 5, or 10 million). (B) Percent (%) difference in GAG matrix within the NP, compared to the untreated ctr, 5 years after treatment with either unstimulated/stimulated discogenic cells (1, 5, or 10 million) or a GDF‐5‐only injection. (C) Predicted central metabolite (glucose, oxygen, and pH) concentrations within the NP of a Grade III intervertebral disc either untreated, subjected to unstimulated/stimulated discogenic cells (1, 5, or 10 million), or a GDF‐5‐only injection. (D) Predicted glucose, oxygen, and pH distribution across a quadrant of a human intervertebral disc, either untreated or subjected to an unstimulated/stimulated 10 million discogenic cell injection or a GDF‐5‐only treatment.