Degenerative Disc Disease (DDD). Current regenerative approaches to degenerative disc disease (DDD) are limited by lack of efficacy, limited sources of raw materials, and cost of production. Fibroblasts are available in ample numbers and are inexpensive to expand in vitro. CybroCell™ is a fibroblast-based universal donor cellular product, which has demonstrated safety and efficacy in preclinical models of DDD. The current study aims to assess safety and efficacy.
It is estimated that approximately 632 million people worldwide are affected by lower back pain, with annual costs in the United States estimated to exceed $100 billion1-3. Degenerative disc disease (DDD), also known as intervertebral disc (IVD) degeneration, is a condition associated with the progressive and irreversible deterioration of one or more of the discs in the spine. Although not all patients with radiological evidence of DDD have lower back pain, DDD is considered one of the major causes of chronic lower back pain4-6.
It is recognized that there are multiple possible and complicated causes of DDD, including genetic, nutritional, and mechanical influences7-9. The processes associated with the degeneration of the disc include a progressive decline in nucleus pulposus (NP) hydration due to the loss of extracellular matrix (ECM) molecules such as aggrecan and collagen10, 11, which is associated in part with reduced oxygenation and increased acidification12. In addition, inflammatory processes such as increased TNF-alpha13-16, macrophage activation17, and NF-kappa B translocation18-22, result in the activation of matrix metalloproteases, which further cause degradation of ECM. Furthermore, these processes result in the apoptosis of cells in the NP, which further results in reduction of ECM synthesis23-25.
This decreased disc hydration results in a loss of mechanical tension in the collagen fibers of the annulus fibrosus and results in abnormal spinal axial loading forces and segmental instability26. Eventually, disc degeneration progresses to cause abnormalities of other components of the disc space, like the endplate and facet joint, which can develop into serious conditions, such as disc herniation, spondylolisthesis, spinal canal stenosis, or facet joint syndrome27-29.
Recent studies have focused on using adult stem cells for disorders such as degenerative disc disease. Mesenchymal stem cells (MSCs) are non-hematopoietic, multipotent progenitor cells that can be isolated from various human adult tissues30. The potential to form cells of multi-lineages has indicated the potential of these cells in cases of degenerative disc disease31. In recent years, MSCs have been shown to possess a broad range of regenerative capabilities, modulating disease progression by repairing lesions closely associated with degenerative disc disease32, 33.
Although MSCs possess various regenerative properties, issues with isolation, expansion, and culturing cost limit their widespread utilization. One potential substitute for MSCs are fibroblasts, which can be readily acquired in large numbers, are relatively easy to expand in vitro, and are economical to produce. Studies show fibroblasts possess a differentiation potential similar to MSC. In one report, mechanical stimulation was applied to dermal fibroblast cells encapsulated in alginate beads using a custom-built bioreactor system for either a 1- or 3-week period at a frequency of 1 Hz for four hours/day under hypoxic conditions. Chondrogenic differentiation of the fibroblasts was observed, as indicated by elevated aggrecan gene expression and an increased collagen production rate34. In vivo ability of fibroblasts to differentiate into chondrogenic cells was demonstrated in a subsequent study. In a recent paper, researchers induced disc degeneration in New Zealand white rabbits by annular puncture and, after four weeks, intradiscally implanted human dermal fibroblasts or saline. Eight weeks after cellular implantation, there was a significant disc height increase in the treated discs compared to control fibroblasts, as well as reduced expression of inflammatory markers, a higher ratio of collagen type II over collagen type I gene expression, and more intense immunohistochemical staining for both collagen types I and II35. An independent group conducted a subsequent study where eight rabbits underwent disc puncture to induce disc degeneration. One month later, cultured fibroblasts, taken from the skin, were injected into the disc. The viability and the potential of the injected cells for reproduction were studied histologically and radiologically. Cellular formations and organizations indicative of histological recovery were observed in the discs to which fibroblasts were transplanted. The histological findings of the discs not transplanted with fibroblasts showed no histological recovery. Radiologically, no finding of improvement was found in both group. However, the fibroblasts injected into the degenerated discs were viable36.
In addition to differentiation into chondrocytic tissues, other studies have shown that fibroblasts can differentiate into other types of cells. In one study, cultured human adult bronchial fibroblast-like cells (Br) were assessed in comparison with mesenchymal cell progenitors isolated from fetal lung (ICIG7) and adult bone marrow (BM212) tissues. Surface immunophenotyping by flow cytometry revealed a similar expression pattern of antigens characteristic of marrow-derived MSCs, including CD34 (-), CD45 (-), CD90/Thy-1 (+), CD73/SH3, SH4 (+), CD105/SH2 (+) and CD166/ALCAM (+) in Br, ICIG7 and BM212 cells. One exception was the STRO-1 antigen, which was only weakly expressed in Br cells. Analysis of cytoskeleton and matrix composition by immunostaining showed that lung and marrow-derived cells homogeneously expressed vimentin and nestin proteins in intermediate filaments. At the same time, they were all devoid of epithelial cytokeratins.
Additionally, alpha-smooth muscle actin was also present in microfilaments of a low number of cells. All cell types predominantly produced collagen and fibronectin extracellular matrix as evidenced by staining with the monoclonal antibodies for collagen prolyl 4-hydroxylase and fibronectin isoforms containing the extradomain (ED)-A together with ED-B in ICIG7 cells. Br cells similar to fetal lung and marrow fibroblasts could differentiate along the three adipogenic, osteogenic and chondrogenic mesenchymal pathways when cultured under appropriate inducible conditions. Altogether, this data indicates that MSCs are present in human adult lungs. They may be actively involved in lung tissue repair under physiological and pathological circumstances37.
Further support for multi-lineage differentiation of fibroblasts used cells isolated from juvenile foreskins—these cells were shown to share a mesenchymal stem cell phenotype and multi-lineage differentiation potential. Specifically, the investigators demonstrated similar expression patterns for CD14(-), CD29(+), CD31(-), CD34(-), CD44(+), CD45(-), CD71(+), CD73/SH3-SH4(+), CD90/Thy-1(+), CD105/SH2(+), CD133(-) and CD166/ALCAM(+) in well-established adipose tissue-derived stem cells and foreskin fibroblastic cells by flow cytometry.
Immunostainings showed fibroblast cells expressed vimentin, fibronectin, and collagen; they were less positive for alpha-smooth muscle actin and nestin, while they were negative for epithelial cytokeratins. Both cell types could differentiate along the adipogenic and osteogenic lineages when cultured under appropriate inducible conditions. Additionally, fibroblasts demonstrated a higher proliferation potential than mesenchymal stem cells. These findings are significant because skin or adipose tissues are easily accessible for cell transplantations in regenerative medicine38. Verification of multi-lineage differentiation of foreskin fibroblasts was provided by a study in which foreskin fibroblasts were demonstrated to possess shorter doubling time than MSC, as well as the ability to multiply more than 50 doublings without undergoing senescence. The cells were positive for the MSC markers CD90, CD105, CD166, CD73, SH3, and SH4 and could be induced to differentiate into osteocytes, adipocytes, neural cells, smooth muscle cells, Schwann-like cells, and hepatocyte-like cells39.