Physicians and surgeons set something like 8 million bone fractures ayear in the U.S. alone. Seven of the 8 million knit uneventfully in amatter of weeks. Kids can cast off their casts sooner than adults.
But the remaining 1 million broken bones cause long-lasting pain andtrouble. For one reason or another, the snapped or splintered shaftsfail to regenerate properly, and often fill with fibrous scarring insteadof healthy bone. A lack of viable bone in the defect site may causeinstability, erosion and infection, all of which can vitiate bone grafts,too.
Three fracture-focused scientists at the University of Michigan, AnnArbor, last July formed a biotech firm, Matrigen Inc., to enlist genetherapy against these peripheral skeletal shortfalls. One of the co-founders, pathologist and molecular geneticist Jeffrey Bonadio, toldBioWorld Today in an interview:
"The university has taken an equity position in Matrigen, in exchangefor exclusive licenses to our technologies." Bonadio is vice-presidentfor research, and sits on the company's board.
Zimmer Inc., of Warsaw, Ind., the orthopedic-implants subsidiary ofBristol-Myers Squibb in New York City, with minority inputs fromthree venture capital groups _ Enterprise Development Fund, AnnArbor; Arch Venture Partners Ltd., Chicago, and FalconTechnologies, Philadelphia _ raised $1.2 million to launchMatrigen, Bonadio said. "We three founders," he added, "are all stillworking out of the university, so our company remains quite virtual."
Bonadio is principal co-author of a paper in today's Proceedings ofthe National Academy of Sciences (PNAS) titled: "Stimulation ofnew bone formation by direct transfer of osteogenic plasmid genes."
The authors' point of departure was that bone _ like liver and skin_ has a substantial capacity for regeneration. "What we thinkhappens," Bonadio explained, "is that osteoprogenitor cells play akey role in fracture repair. They appear on the wound scene incoordination with the repair fibroblasts in capillary blood vessels.
From Osteogenic Theory To Practice
"Osteogenic stem cells, too, are thought to be present, and when awound _ in our case, a fracture _ is created, it galvanizes them intoaction."
To recreate this action in rats, the Michiganders constructed threerecombinant-cDNA expression plasmids, powered by a vector. Thefirst encoded bone morphogenic proteins (BMP), which are normallyexpressed by fetal osteoblasts. Recombinant BMPs initiate boneprogenitor differentiation. The second vehicle encoded the businessend (amino-acids 1 through 34) of human parathyroid hormone(hPTH), a co-factor in bone formation. The third combined both theBMP and PTH genes.
From 50 anesthetized rats (a preferred animal model for orthopedicresearch) the team excised segments of femur 5 mm (3/16") long, byabout 2mm in diameter. Into these gaps they placed matrices ofcollagen sponge soaked in one or another of the three plasmids.
To hold the two loose stumps of midshaft bone in position, Bonadioand his co-authors inserted four pins into holes drilled on either sideof the gap. "The pins stuck up through the skin," he said, "with aplate applied across them outside the skin _ what the orthopedicworld calls an external fixator." Besides immobilizing theexperimental site, Bonadio said, "It was also humane, in the sensethat the animals are pain-free when they have this form ofstabilization. They wake up and walk, right after surgery."
Two of the rats kept right on walking; they remained ambulatory for20 weeks following the experiment, which was conducted between1994 and July 1995.
The box score of trial results reported that all 14 of the rats whoreceived the gene therapy grew new bone, while 36 control animals,left to heal on their own, acquired fibrous scarring instead.
Significantly, animals that received either plasmid BMP or PTH tooknine weeks to bridge the bony gap, whereas those who got thecombined-dose plasmid healed in a matter of four weeks.
"If you were to take DNA in solution, like naked-DNA technology,for example, and drip that into the void in the femur, it woulddissipate; there's nothing to hold it there. So one function of ourmatrix is to hold the DNA in the void, so the cells can take it up andexpress the recombinant protein."
He went on, "In our particular case, our target cell is the granulationfibroblast. It will naturally grow into and fill a void, but we think thatthe matrix in and of itself will act as a scaffolding that facilitates theprocess, which we sometimes call `baiting the trap.'"
Bonadio described his team's successful matrix-delivered constructas a "sustained release" of the therapeutic genes. "In treatingfractures," he said, "there's an imperative to provide a fix that isimmediate. Somebody comes in with a broken bone, you're not goingto wait 24 or 48 hours for granulation-tissue fibroblasts to appear onthe scene before you provide them with a therapy."
Coating Medical Devices With DNA
Ahead of Matrigen lie two new developments: "One is that we'retrying to extend these studies now to a dog model. That's the nextstep in our pathway. And the second point to the advantage of asynthetic sustained-release preparation as opposed to a collagensponge."
So the team is perfecting a method to emulsion-coat medical deviceswith sustained-release plasmid DNA.
They already have tested coated surgical sutures in vivo in ratmuscle, and, Bonadio said, "we know we can coat orthopedictitanium screws and stainless-steel plate. Our feeling," he added, "isthat if we can coat those things, we can coat just about anything."
So far, Matrigen has concluded two corporate research partnerships.One, Bonadio said, is with Parke-Davis, a division of Warner-Lambert Co., at its pharmaceutical research facility in Ann Arbor. "Itis for using our sustained-release technology to develop a localizeddelivery system for one of their drugs."
The other collaboration, dealing with Matrigen's bone program, iswith Zimmer. "We always said to ourselves," Bonadio said, "if wewere successful in healing bone, then this gene-transfer technologycould be iterated to clinical applications in other tissues and organs."The process of wound-healing, he said, "is stereotyped, in terms ofthe cellular sequence of events that occurs. It's the same for liver,lung, heart, tendon, ligament _ in fact, every organ and tissue in thebody."
That sequence starts with bleeding, which the body checks byclotting. When a clot has served its purpose, it is absorbed, leavingwhat Bonadio calls "a tissue void." Nature, he said, "abhors such avoid, and fills it with granulation tissue; that is, fibroblasts _ ourtarget for gene transfer." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.