Fat-derived cells Enhance the Bone-Forming Capacity of Hypertrophic Cartilage Matrices


Treating particular bone defects or injuries present a substantial challenges for clinicians. The method of choice usually involves the use of an “autologous” bone graft (“autologous” simply means that the graft comes from the patient’s own bone). However, autologous bone grafts have plenty of limitations. For example, if a patient has a large enough bone defect, there is no way the orthopedist and take bone from a donor site without causing a good deal of risk to the donor site. Even with small bone grafts, so-called “donor site morbidity” remains a risk. Having said that, plenty of patients have had autologous bone grafts that have worked well, but larger bone injuries or defects are not treatable with autologous bone grafts.

The answer: bone substitute materials. Bone substitute materials include tricalcium phosphate, hydroxyapatite, cement, ceramics, bioglass, hydrogels, polylactides, PMMA or poly(methy methacrylate) and other acrylates,, and a cadre of commercially available granules, blocks, pastes, cements, and membranes. Some of these materials are experimental, but others do work, even if do not work every time. The main problem with bone substitute materials is that, well, they are not bone, and, therefore lack the intrinsic ability to induce the growth of new bone (so-called osteoinductive potential) and their ability to integrate into new bone is also a problem at times.

We must admit that a good deal of progress has been made in this area and it’s a good thing too. Many of our fabulous men and women-at-arms have returned home with severe injuries from explosives and wounds from large-caliber weapons that have shattered their bones. These courageous men and women have been the recipient of these technologies. However, the clinician is sometimes left asking herself, “can we do better?”

A new paper from the laboratories of Ivan Martin and Claude Jaquiery from the University Hospital of Basel suggests that we can. This paper appeared in Stem Cells Translational Medicine and describes the use of a hypertrophic cartilage matrix that was seeded with cells derived from the stromal vascular faction of fat to not only make bone in the laboratory, but to also heal skull defects in laboratory animals. While this work benefitted laboratory animals, it was performed with human cells and materials, which suggests that this technique, if it can be efficiently and cheaply scaled up, might be usable in human patients.
The two lead authors of this paper, Atanas Todorov and Matthias Kreutz and their colleagues made hypertrophic cartilage matrices from human bone marrow mesenchymal stem cells (from human donors) that were induced to make cartilage. Fortunately, protocols have been very well worked out and making cartilage plugs with chondrocytes that are enlarged (hypertrophic) is something that has been successfully done in many laboratories. After growing the mesenchymal stem cells in culture, half a million cells were induced to form cartilage with dexamethasone, ascorbic acid 2-phosphate, and the growth factor TGF-beta1. After three weeks, the cartilage plugs were subjected to hypertrophic medium, which causes the cartilage cells to enlarge.

Chondrocyte enlargement is a prolegomena to the formation of bone and during development, many of our long bones (femur, humerus, fibula, radius, etc.), initially form as cartilage exemplars that are replaced by bone as the chondrocytes enlarge. Ossification begins when a hollow cylinder forms in the center of the bone (known as the periosteal collar). The underlying chondrocytes degenerate and die, thus releasing the matrix upon which calcium phosphate crystals accrete. The primary ossification center commences with the calcification of the central shaft of the bone and erosion of the matrix by the invasion of a blood vessel. The blood vessels bring osteoprogenitor cells that differentiate into osteoblasts and begin to deposit the bone matrix.

Next, Todorov and his crew isolated the stromal vascular fraction from fat that was donated by 12 volunteers who had fat taken from them by means of liposuction. The fat is then minced, digested with enzymes, centrifuged, filtered and then counted. This remaining fraction is called the stromal vascular fraction or SVF, and it consists of a pastiche of blood vessel-forming cells, mesenchymal stem cells, and bone-forming cells (and probably a few other cells types too). These SVF cells were seeded onto the hypertrophic cartilage plugs and used for the experiments in this paper.

First, the SVF-seeded plugs were used to grow bone in laboratory rodents. The cartilage plugs were implanted into the backs for nude mice. Different cartilage plugs were used that had been seeded with gradually increasing number of SVF cells. The implanted plugs definitely made ectopic bone, but the amount of bone they made was directly proportional to the number of SVF cells with which they had been seeded. Staining experimental also showed that some of the newly-grown bone came from the implanted SVF cells.

Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.
Ectopic bone formation. Grafts based on devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction cells from adipose tissue and implanted subcutaneously in nude mice. (A): Representative hematoxylin and eosin, Masson-Tri-Chrome, and Safranin-O (Saf-O) staining and in situ hybridization for human ALU sequences (dark blue = positive) after 12 weeks in vivo. Saf-O stainings are blue-green because of lack of glycosaminoglycans and counterstaining with fast green. Osteoid matrix and bone marrow are visible. Scale bars = 200 µm. (B): Stainings for metalloproteinase (MMP)13 and MMP9, as well as for the N-terminal neoepitope at the major MMP cleavage site (DIPEN) after 12 weeks in vivo (red/pink = positive). Scale bars = 50 µm. +, osteoid matrix; ⋆, bone marrow. Abbreviations: ALU, Arthrobacter luteus; H&E, hematoxylin and eosin; Masson, Masson’s trichrome; MMP, metalloproteinase; Saf-O, Safranin-O; SVF, stromal vascular fraction.

In the second experiment, Todorov and Kreutz used these SVF-seeded cartilage plugs to repair skull lesions in rats. Once again, the quantity of bone produced was directly proportional to the number of SVFs seeded into the cartilage matrices prior to implantation. In both experiments, the density of SVF cells positively correlates with the bone-forming cells in the grafts and the percentage of SVF-derived blood vessel-forming cells correlates with the amount of deposited mineralized matrix.

Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.
Bone repair capacity. Devitalized hypertrophic cartilage pellets were embedded in fibrin gel without or with stromal vascular fraction (SVF) cells from adipose tissue and implanted in rat calvarial defects. (A): Mineralized volume quantified by microtomography (n = 9 grafts assessed per group). (B): Bone area assessed in histological sections, expressed as percentage of total defect area (n = at least 24 sections assessed per group). ∗∗∗∗, p < .0001. (C, D): Representative three-dimensional microtomography reconstructions (left) and hematoxylin/eosin (H&E) staining (right) of the calvarial defects filled with devitalized grafts, implanted without (C) or with (D) activation by SVF cells after 4 weeks. Dotted circles indicate the defect borders (4 mm diameter). Scale bars = 500 µm. (E): Microtomography (left) and H&E staining (middle and right) displaying the bridging between hypertrophic matrix and bone of the calvarium, or the fusion of single pellets (right) in activated grafts. White bar = 850 µm; black bars = 500 µm. Dotted lines indicate the edge of the calvarium. (F): In situ hybridization for Arthrobacter luteus sequences showing the presence of human cells (dark blue, positive) in the explants. Scale bar = 200 µm. Abbreviations: C, calvarium; dev, fibrin gel without stromal vascular fraction; dev + SVF, fibrin gel with stromal vascular fraction; P, hypertrophic matrix; SVF, stromal vascular fraction.

This is not the first time scientists have proposed the use of cartilage plugs to induce the formation of new bone. Van der Stok and others and Bahney and colleagues were able to repair segmental bone defects in laboratory rodents. Is this technique transferable to human patients? Possibly. Hypertrophic cartilage is relatively easy to make and it is completely conceivable that hypertrophic cartilage wedges can be sold as “off-the-shelf” products for bone treatments. SVF can also be derived from the patient or can be derived from donors.

Furthermore, the protocols in this paper all used human cells and grew the products in immunodeficient rats and mice. Therefore, in addition to scaling this process up, we have a potentially useful protocol that might very well be adaptable to the clinic.

The efficacy of this technique must be confirmed in larger animal model system before human trials can be considered. Hopefully human trials are in the future for this fascinating technique.

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Published by

mburatov

Professor of Biochemistry at Spring Arbor University (SAU) in Spring Arbor, MI. Have been at SAU since 1999. Author of The Stem Cell Epistles. Before that I was a postdoctoral research fellow at the University of Pennsylvania in Philadelphia, PA (1997-1999), and Sussex University, Falmer, UK (1994-1997). I studied Cell and Developmental Biology at UC Irvine (PhD 1994), and Microbiology at UC Davis (MA 1986, BS 1984).

2 thoughts on “Fat-derived cells Enhance the Bone-Forming Capacity of Hypertrophic Cartilage Matrices”

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