**7.2.2 Histologic findings**

Under H&E staining, scar tissue was present in group A, which was characterized by a disordered arrangement of fibroblasts in an edematous stroma (Fig 9). The interface of scar tissue with subchondral bone was marked by the presence of dilated capillaries and venules. In group B scar tissue was less pronounced, and islands of hyaline-like cartilage were seen at the interface with subchondral bone and also adjacent to normal cartilage at the defect margins. Edema was also less marked than in the control group. In group C there was chondrogenesis with evidence of hyaline cartilage formation. The hyaline cartilage also showed features of maturation as evidenced by a linear arrangement of chondrocytes extending from the subchondral bone toward the surface. No edema was seen in this group. With Safranin-O staining, proteoglycans were notably absent from the repair tissue in group A. In group B proteoglycans were seen only at the base and sides of the defect in the same distribution as the hyaline-like cartilage. In group C there was marked proteoglycan accumulation in the deeper layers, excluding the perichondrium, which normally does not contain proteoglycans.

Articular Cartilage Regeneration with Stem Cells 141

The combination of HA and MA in group C yielded the best results in that the repair tissue was composed of true hyaline cartilage, showing vertical orientation of chondrocyte nests and the presence of type II collagen and proteoglycans in the intermediate and deep cartilage layers, with type I collagen confined to the superficial layer and perichondrium (Fig 9). This suggests that the combination of HA and MA is most effective in neutralizing the paracrine factors, and its effects persist for the duration of the repair process. One of the essential active components in MA is likely the MSC content, given that a previous study in a porcine model showed that cultured autologous MSCs injected intraarticularly together

with HA could produce the same results as in the group C animals (Lee et al, 2007).

**7.2.4 Conclusion on the animal model** 

drilling resulted in a better cartilage repair.

**7.3 Clinical trial** 

**7.2.5 Clinical relevance from the animal model** 

regeneration was possible with this novel approach.

**7.3.1 Patient selection – indication for surgery** 

the following sections (Saw et al, 2011).

The presence of edema in the repair tissue appears to be proportional to the degree of fibrous scarring and was most marked in group A animals. The dilated capillaries and venules at the base of the defect suggest an inflammatory process that originates in the subchondral bone. These observations indicate that the paracrine factors, which promote fibrous tissue formation, are closely related to the inflammatory process that occurs after mechanical disruption of the chondral plate and that both HA and MA may be able to suppress inflammation locally.

This preclinical experimental study in the goat model concluded that postoperative intraarticular injections of autologous MA in combination with HA after subchondral

After arthroscopic subchondral drilling, postoperative intraarticular injections of autologous progenitor cells in combination with HA may result in better articular cartilage regeneration.

A human clinical trial followed the preclinical animal studies. The surgical technique applied in the clinical trial involved standard marrow stimulation in the form of arthroscopic subchondral drilling and postoperative intraarticular injections of autologous peripheral blood progenitor cells (PBPC) in combination with HA. The objective of the trial was to assess whether the results of the preclinical animal model could be replicated in the human knee joint. The purpose of the clinical trial was to evaluate the quality of resultant articular cartilage regeneration. A hypothesis was made that articular hyaline cartilage

The early results with histology were published in *Arthroscopy: The Journal of Arthroscopic and Related Surgery, Vol 27, No 4 (April), 2011: pp 493-506* . An excerpt of the paper is presented in

The diagnosis of chondral injury was made after clinical and radiologic evaluation. Chondral lesions were graded according to the International Cartilage Repair Society (ICRS) Cartilage Injury Evaluation Package (Brittberg & Peterson, 1998) The inclusion criteria were patients with ICRS grade III and IV lesions, defects of any size and number, age 18 to 60 years, deformity (lateral patella maltracking or axis correction) correctable at the time of surgery, and ligamentous instability deemed reconstructable at the same time. The exclusion criteria were patients with disease progression such that total knee arthroplasty was indicated; a history of

Under collagen staining, the scar tissue in group A was found to contain only type I collagen, with an absence of staining for type II collagen. In group B type I collagen staining in the repair tissue was less pronounced, with light staining for type II collagen around the areas of hyaline-like cartilage. In group C type I collagen staining was found only in the perichondrium, whereas the deeper cartilage stained strongly for type II collagen.


Fig. 9. Macroscopic and histologic findings for representative subjects from all 3 groups. (N, normal cartilage; R, repair cartilage; V, dilated capillaries and venules; I, islands of hyalinelike cartilage).

#### **7.2.3 Discussion on the animal model**

Penetration of the subchondral bone was shown to release the underlying marrow, which initiated repair of the chondral defect with fibrocartilage in a pattern that was observed in group A animals. The reason why fibrous tissue forms instead of hyaline cartilage is not known, but it is likely that the local microenvironment contains paracrine factors that either promote fibrous tissue formation, inhibit cartilage growth, or both.

The addition of HA in group B animals improved the quality of repair tissue by allowing hyaline-like cartilage to form. This suggests that HA modifies the microenvironment in such a way that neutralizes these paracrine factors. The incomplete regeneration could be because of HA being only partially effective or the duration of administration being too short. It appeared that the region adjacent to subchondral bone and the interface with normal cartilage seemed to be most favorable to cartilage formation.

Under collagen staining, the scar tissue in group A was found to contain only type I collagen, with an absence of staining for type II collagen. In group B type I collagen staining in the repair tissue was less pronounced, with light staining for type II collagen around the areas of hyaline-like cartilage. In group C type I collagen staining was found only in the

Fig. 9. Macroscopic and histologic findings for representative subjects from all 3 groups. (N, normal cartilage; R, repair cartilage; V, dilated capillaries and venules; I, islands of hyaline-

Penetration of the subchondral bone was shown to release the underlying marrow, which initiated repair of the chondral defect with fibrocartilage in a pattern that was observed in group A animals. The reason why fibrous tissue forms instead of hyaline cartilage is not known, but it is likely that the local microenvironment contains paracrine factors that either

The addition of HA in group B animals improved the quality of repair tissue by allowing hyaline-like cartilage to form. This suggests that HA modifies the microenvironment in such a way that neutralizes these paracrine factors. The incomplete regeneration could be because of HA being only partially effective or the duration of administration being too short. It appeared that the region adjacent to subchondral bone and the interface with normal

promote fibrous tissue formation, inhibit cartilage growth, or both.

cartilage seemed to be most favorable to cartilage formation.

like cartilage).

**7.2.3 Discussion on the animal model** 

perichondrium, whereas the deeper cartilage stained strongly for type II collagen.

The combination of HA and MA in group C yielded the best results in that the repair tissue was composed of true hyaline cartilage, showing vertical orientation of chondrocyte nests and the presence of type II collagen and proteoglycans in the intermediate and deep cartilage layers, with type I collagen confined to the superficial layer and perichondrium (Fig 9). This suggests that the combination of HA and MA is most effective in neutralizing the paracrine factors, and its effects persist for the duration of the repair process. One of the essential active components in MA is likely the MSC content, given that a previous study in a porcine model showed that cultured autologous MSCs injected intraarticularly together with HA could produce the same results as in the group C animals (Lee et al, 2007).

The presence of edema in the repair tissue appears to be proportional to the degree of fibrous scarring and was most marked in group A animals. The dilated capillaries and venules at the base of the defect suggest an inflammatory process that originates in the subchondral bone. These observations indicate that the paracrine factors, which promote fibrous tissue formation, are closely related to the inflammatory process that occurs after mechanical disruption of the chondral plate and that both HA and MA may be able to suppress inflammation locally.

## **7.2.4 Conclusion on the animal model**

This preclinical experimental study in the goat model concluded that postoperative intraarticular injections of autologous MA in combination with HA after subchondral drilling resulted in a better cartilage repair.
