Sports Medicine Research Laboratory

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		Molecular Orthopedics
		Platelet Characterization
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		Joint Imaging
		Biomechanics of Injury Repair
		Histology/ Immunohistochemistry
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		Injury Prevention
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Histology and Immunohistochemistry

Team Leader:	Lena Liu Department of Pathology Children's Hospital Boston
Collaborator:	Lillian B. Nanney, Ph.D. Professor of Plastic Surgery, and Professor of Cell and Developmental Biology Vanderbilt University Medical Center
Team	Eduardo Abreu, PhD, Tyler Welch, MD, Percy Ballard, MD
Funding:	OREF

In collaboration with Lena Liu in the Department of Pathology at Children's Hospital Boston, and Dr. Lillian Nanney in the Department of Cell Biology at Vanderbilt University Medical Center, we are actively using histologic and immunohistochemical analysis to help us better understand the cellular and tissue level processes associated with the injury and repair responses of joint tissues.

Defining Natural History of Response to Injury of Joint Tissues

Our early work focused on the systematic histologic and pathologic examination of injured joint tissues, including the anterior cruciate ligament (ACL), meniscus and articular cartilage.

To identify and define the deficiencies in the healing process for these tissues, IRB approval was obtained, and intact and injured human specimens of intra-articular tissues were collected over a 5 year period. Evaluated tissues included ligament, meniscus, cartilage and tendon.

Gross examination and histomorphometric analyses was performed in a consistent manner for all tissues. Parameters of cell density, vascularity and extracellular matrix organization were systematically evaluated. Cellular functions including proliferative capability, migration characteristics and capacity for extracellular matrix production were evaluated for cells from normal(3, 5) and injured tissues(6, 10).

Fig. 1: Histologic response of the human ACL to rupture.
A. Histologic appearance of the normal ACL showing fibroblasts (blue nuclei; 40X).
B. Histologic appearance of ACL tissue 3 months after rupture showing increased cell density in the ligament ends (40X).
C. Area of increased capillary density in ACL ends 3 months after rupture (20X).
D. Synovial layer which has reformed over the ligament ends at 8 weeks after rupture (BV = blood vessel, 40X).
Sections are immunohistochemistry for alpha-smooth muscle actin (SMA) where red demonstrates a positive stain for SMA, with a blue counter-stain for cell nuclei.

Fig. 2
Click for larger image

By comparing the tissues in the pre- and post-injury state, we identified an intrinsic cell and vascular response adjacent to the defect(4) (Fig. 1B and 1C), as well as a remarkable capability for cells from these tissues to proliferate and migrate in vitro(3, 6, 10). Even the articular chondrocytes from patients with end-stage osteoarthritis had this capacity(10). However, at no time point was any filling of the injury site observed (Fig. 2A and C). This was true for cartilage(8), meniscus(2) and the anterior cruciate ligament(4).

Summary of Findings

These findings of a productive cellular response to injury for joint tissues, but a lack of any filling of the wound site led us to our novel hypothesis: that it is the premature failure of the provisional scaffold which prevents healing of tissues inside joints.

Testing The Provisional Scaffold Hypothesis

In this pilot experiment, six animals had venipuncture to withdraw 4.5 cc of blood (for preparation of the autologous PRP gel) and subsequently underwent bilateral arthrotomies and partial transection of the ACL.

For each animal, one ACL was left untreated, while the second knee had a collagen- platelet-rich plasma hydrogel placed into the defect.

Ten days after implantation, dense, collagenous tissue was forming at the border of the ACL and the collagen-PRP hydrogel, and fibroblasts and vessels were noted in the bulk of the collagen-PRP device.

By three weeks, additional capillary invasion and hypercellular scar formation was noted in the defect in the area where the collagen-PRP gel had been placed (Fig. 3C).

The appearance of this scar tissue was similar to that seen in the control MCL (Fig. 3B). In contrast, in the control knee, no tissue was noted to form in the gap of the transected ACL even at three weeks (Fig. 3A)(7).

Fig. 3
Click for larger image

This work has shown that delivery of the appropriate bridge can stimulate the intrinsic healing response for tissues inside joints. This bridge can then also be used as a foundation for the delivery of cytokine and cell-based treatments. Stimulation of healing of tissues inside the joint could significantly alter the way we approach these injuries.

In collaboration with Dr. Lillian Nanney at Vanderbilt University Medical Center we have also been actively using immunohistochemistry to identify cellular behaviors within and around the wound site. For example, we have found that fibrin is still present within the wound site of an ACL defect treated with a collagen-PRP scaffold as far as six weeks out from injury(7).

Fig. 4: The untreated ACL defect remains open and empty, but the treated ACL defect and healing patellar tendon defect are both filled with a fibrin rich scaffolding material (Immunohistochemistry where brown represents a positive stain).


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