## 2 Materials and Methods
### Surgical Protocol  
The animal surgical protocol for this study was reviewed and approved by the Ibex Preclinical Research Institutional Animal Care and Use Committee (IACUC). The protocol number was C12611-Ibex-09-10 and approved on 10 April 2013. All animal work, histology, and radiographic analysis were completed by WuXi AppTec (St. Paul, MN, USA) unless otherwise stated. A total of 30 adult, male, New Zealand white rabbits were bilaterally implanted with grafts of porous and non-porous HA granules into a critical size defect created on the femoral condyle. First, bone marrow aspirate was harvested from the iliac crests of each animal (2 cc per rabbit). The porous and non-porous HA grafts were separately mixed with the autologous bone marrow aspirate (BMA) of each animal  
Figure 1: Porous hydroxyapatite (HA) granules (**A**) present significantly greater surface area per volume than traditional, non-porous microparticles (**B**); while the surface topography based on the 500–800 nm size of pre-sintered HA particles is similar for the porous (**C**) and non-porous (**D**) granules. (**E**) Mesenchymal cells proliferate throughout the porous HA scaffold in vitro observed by staining for nuclei (blue, DAPI) and fibrillar actin (red, rhodamine phalloidin). (**F**) The porous (**left**) and non-porous (**right**) HA graft materials form a moldable putty when hydrated with bone marrow aspirate. prior to implantation by combining 0.5 cc BMA with 0.5 cc porous or non-porous HA granules (1:1 ratio by volume), respectively. Each biomaterial contained a gelatin carrier that dissolved when wetted with BMA and formed a viscous gel around each HA particle. The resulting putty was loaded into 1 mL syringes, with the distal tip removed in order to form bone graft logs for implantation. The excess BMA was transported to the laboratory (Celling Biosciences, Austin, TX, USA) for cell analysis. Two drill defects (approximately 6 mm in diameter and 8-10 mm in length) were created in each animal--one in each femoral condyle--and were filled with porous HA graft on the left side and non-porous HA graft on the right side (Figure 2). Approximately 0.25 cm\({}^{3}\) of hydrated graft material was implanted in each defect by pushing the putty out of the 1 mL syringe. Each animal received sufficient analgesic (butophanol 0.5-1.0 mg/kg) injection prior to and after the surgery. The animals also received a fentanyl transdermal patch on the skin for additional analgesia up to 3 days. Ten animals were implanted per time point (4, 8, and 13 weeks). Radiographs were collected post-operatively and at termination for each animal. At necropsy, 4, 8, or 13 weeks after implantation, the randomly assigned 10 animals were euthanized, and gross observations of the implant sites were observed and recorded. Implantation sites were excised and placed in 10% neutral buffered formalin for fixation. Radiographic microCT analysis was performed on the 8- and 13-week animals (Numira Biosciences, Inc., Salt Lake City, UT, USA). Histological preparation was done immediately after animal sacrifice for the 4-week samples, and following microCT analysis on the 8- and 13-week samples. Standard techniques were used to prepare slides for histological analysis. This included decalcification, paraffin embedding, microtome sectioning, and staining. Two slides were prepared from each defect, one at the central level of the drill defect and one from the interface with original bone. The slides were stained with hematoxylin/eosin (H&E). Each section was analyzed by a pathologist for local tissue reaction, following the ISO 10993-6 guidelines--first by an independent pathologist (WuXi ApTec, St. Paul, MN, USA), and reviewed by a second independent pathologist (NAMSA, Minneapolis, MN, USA). Figure 2: At the onset of surgery, bone marrow aspirate was drawn from the iliac crest of each rabbit (**A**) and mixed with porous or non-porous hydroxyapatite granule at 1:1 ratio by volume in a 1 mL syringe (**B**) to form implantable cylinders (**C**).